Malaria antigens and methods of use

ABSTRACT

The invention is directed to a composition comprising one or more polypeptides or one or more nucleic acid sequences that can induce a protective immune response against  Plasmodium  species that infect humans. The invention also is directed to a method of using such compositions to induce a protective immune response against a  Plasmodium  parasite in a mammal.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with Government support under Grant Number1R43AQ1084269-01 awarded by the National Institute of Allergy andInfectious Diseases (NIAID). The Government has certain rights in thisinvention.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: One 828,208 Byte ASCII (Text) file named“720344SequenceListing_ST25” created on Nov. 21, 2016

BACKGROUND OF THE INVENTION

Malaria is one of the most devastating parasitic diseases affectinghumans. The Centers for Disease Control (CDC) estimate that over threebillion people live in areas at risk of malaria transmission in 106countries and territories (e.g., parts of Africa, Asia, the Middle East,Central and South America, Hispaniola, and Oceania). The World HealthOrganization (WHO) and CDC also estimate that in 2010, malaria causedover 200 million clinical episodes 655,000 deaths, with the majority ofdeaths occurring in Africa. About 86% of the malaria deaths in 2010occurred in children. On average, 1,500 cases of malaria are reportedannually in the United States, and malaria is a major health concern toU.S. military personnel deployed to tropical regions of the world. Forexample, in August 2003, 28% of the 26th Marine Expeditionary Unit andJoint Task Force briefly deployed to Monrovia, Liberia, were infectedwith the malaria parasite Plasmodium falciparum. In addition, one157-man Marine Expeditionary Unit sustained a 44% malaria casualty rateover a 12-day period while stationed at Robert International Airport inMonrovia. In all conflicts during the past century conducted in malariaendemic areas, malaria has been the leading cause of casualties,exceeding enemy-inflicted casualties in its impact on “person-days” lostfrom duty.

To combat malaria during U.S. military operations, preventive drugs,insect repellants, and barriers have been used with some success, butdeveloping drug resistance by the malaria parasite and insecticideresistance by mosquito vectors has limited the efficacy of these agents.Moreover, the logistical burden and side effects associated with the useof these agents often is associated with high non-compliance rates.Vaccines are the most cost effective and efficient therapeuticinterventions for infectious diseases. In this regard, vaccination hasthe advantage of administration prior to military deployment and likelyreduction in non-compliance risks. However, decades of research anddevelopment directed to a malaria vaccine have not proven successful.Recent efforts have focused on developing vaccines against severalspecific malaria genes and delivery vector systems including adenovirus,poxvirus, and plasmids. The current status of malaria vaccinedevelopment and clinical trials is reviewed in, for example, Vaughan etal., Curr. Opin., Immunol., 24(3): 324-331 (2012); Schwartz et al.,Malaria Journal, 11: 11 (2012); Tyagi et al., J. Control Release,162(1): 242-254 (2012); Graves and Gelband, Cochrane Database Syst.Rev., 1: CD000129 (2003); Moore et al., Lancet Infect. Dis., 2: 737-743(2002); Carvalho et al., Scand. J. Immunol., 56: 327-343 (2002); Moorthyand Hill, Br. Med. Bull., 62: 59-72 (2002); Greenwood and Alonso, Chem.Immunol., 80: 366-395 (2002); and Richie and Saul, Nature, 415: 694-701(2002).

An unprecedented quantity of genomic data has emerged from thesequencing and functional genomic analysis of many disease-causingorganisms, including malaria. Indeed, it has been determined that theparasite Plasmodium falciparum encodes an estimated 5,268 putativeproteins (see Gardner et al., Nature, 419: 498-511 (2002)). This geneticinformation can be exploited for the systematic discovery of novelantigens for vaccine development. In the past, target antigens forgenetic vaccines have been identified based mainly on their abundance inthe pathogen of interest and their susceptibility to neutralization byantibodies generated in infected individuals and animal models. Thisapproach has failed to yield effective vaccines against many of the mostdevastating infectious diseases.

With regard to malaria, less than 5% of the Plasmodium falciparum genomeis represented by antigens currently in clinical development. However, anumber of potential vaccine candidates targeted againstpre-erythrocytic, erythrocytic and sexual stages of P. falciparum areunder various stages of clinical development (see, e.g., Crompton etal., J. Clin. Invest., 120: 4168-4178 (2010)). The RTS,S vaccine is themost clinically advance malaria vaccine. RTS,S is a pre-erythrocyticstage vaccine based on the P. falciparumcircumsporozoite protein (CSP),and provides protective efficacy in phase II clinical trials of 30-50%against pathogen challenge (see, e.g., Cesares et al., Vaccine, 28:4880-4894 (2010); Stoute et al., N Engl. J. Med., 336: 86-91 (1997);Kester et al., Vaccine, 26: 2191-2202 (2008); Kester et al., J. Infect.Dis., 200: 337-346 (2009); Kester et al., Vaccine, 25: 5359-5366 (2007);and Zeeshan et al., PLoS ONE, 7(8): e43430 (2012)). Initial results ofphase III clinical trials show that the RTS,S vaccine providesprotective efficacies of 56% and 47% against clinical and severemalaria, respectively, in African children age 5 to 17 months (see,e.g., Agnandji et al., N Engl. J. Med., 365: 1863-1875 (2011)). Theprotection afforded by this protein-based vaccine, however, is shortlived (3-8 weeks).

Other recent efforts at developing a malaria vaccine have focused onseveral specific genes and their delivery using various different vectorsystems including adenovirus, poxvirus, and plasmid DNA. It is notapparent, however, whether these recombinant vaccines are effectiveagainst malaria, or if they encode the most potent protective antigens.It is clear that protective antigens do exist for the malaria pathogenPlasmodium falciparum, as evidenced by the ability of irradiatedsporozoites to induce cellular immune responses in human subjects androbust sterile protection against parasite challenge (see, e.g.,Nussenzweig and Nussenzweig, Adv. Immunol., 45: 283-334 (1989), andHoffinan et al., J. Infect. Dis., 185: 1155-1164 (2002)).

Thus, there remains a need for compositions containing improved antigensthat induce potent protective immunity against challenge withmalaria-causing parasites. The invention provides such a composition.This and other advantages of the invention will become apparent from thedetailed description provided herein.

BRIEF SUMMARY OF THE INVENTION

The invention provides a composition comprising a pharmaceuticallyacceptable carrier and one or more isolated polypeptides, wherein eachof the one or more isolated polypeptides comprises (a) the amino acidsequence of KKERYXIWXRXPKXELHCHLD (SEQ ID NO: 1), wherein X is a lysine(K) residue, a glutamic acid (E) residue, an arginine (R) residue, anisoleucine (I) residue, a leucine (L) residue, a cysteine (C) residue,or a valine (V) residue, (b) the amino acid sequence of KYKEGVVLMEFRYSP(SEQ ID NO: 2), (c) an amino acid sequence comprising at least 20contiguous amino acid residues of the sequence EDLAKXAVXXKYKEGVVLMEFRYSP(SEQ ID NO: 3), wherein X is a histidine (H) residue, a tryptophan (W)residue, a phenylalanine (F) residue, an isoleucine (I) residue, anasparagine (N) residue, or a glutamic acid (E) residue, or (d) an aminoacid sequence comprising at least 20 contiguous amino acid residues ofthe sequence KSMDTHPIRXLYDAGVKVSVNSDDPGMFL (SEQ ID NO: 4), wherein X isa glutamine (Q) residue, a methionine (M) residue, or a lysine (K)residue, and wherein each of the one or more isolated polypeptidesinduces a protective immune response against Plasmodium falciparumand/or Plasmodium vivax in a mammal.

The invention provides a composition comprising a pharmaceuticallyacceptable carrier and one or more isolated polypeptides, wherein eachof the one or more isolated polypeptides comprises (a) an amino acidsequence comprising at least 20 contiguous amino acid residues of thesequence MKKDREPIDEDEMRITSTGRMTNYVNYGAKILG (SEQ ID NO: 20), (b) an aminoacid sequence comprising at least 20 contiguous amino acid residues ofthe sequence KIKATGNAIGKAVTLAEIIKRRFKGLHQIT (SEQ ID NO: 21), or (c) anamino acid sequence comprising SEQ ID NO: 22, and wherein each of theone or more isolated polypeptides induces a protective immune responseagainst Plasmodium falciparum and/or Plasmodium vivax in a mammal.

The invention provides a composition comprising a pharmaceuticallyacceptable carrier and one or more isolated polypeptides, wherein eachof the one or more isolated polypeptides comprises (a) an amino acidsequence which comprises at least 40 contiguous amino acid residues ofthe sequenceMGKEKTHINLVVIGHVDSGKSTTTGHIIYKLGGIDRRTIEKFEKESAEMGKGSFKYAWVLDKLKAERERGITIDIALWKFETPRYFFTVIDAPGHKDFIKNMITGTSQADVALLVVPAEVGGFEGAFSKEGQTKEHALLAFTLGVKQIVVGVNKMDTVKYSEDRYEEIKKEV (SEQ ID NO: 30), (b) an amino acid sequence which comprises at least55 contiguous amino acid residues of the sequenceDYLKKVGYQADKVDFIPISGFEGDNLIEKSDKTPWYKGRTLIEALDTMEPPKRPYDKPLRIPLQGVYKIGGIGTVPVGRVETGILKAGMVLNFAPSAVVSECKSVEMHKEVLEEARPGDNIGFNVKNVSVKEIKRGYVASDTKNEPAKGCSKFTAQVIILNHPGEIKNGY(SEQ ID NO: 31), (c) the amino acid sequence ofHISCKFLNIDSKIDKRSGKVVEENPK (SEQ ID NO: 32), or (d) an amino acidsequence which comprises at least 20 contiguous amino acid residues ofthe sequence LEPKKPMVVETFTEYPPLGRFAIRDMRQTIAVGIIK (SEQ ID NO: 33), andwherein each of the one or more isolated polypeptides induces aprotective immune response against Plasmodium falciparum and/orPlasmodium vivax in a mammal.

The invention provides a composition comprising a pharmaceuticallyacceptable carrier and one or more isolated polypeptides, wherein eachof the one or more isolated polypeptides comprises (a) the amino acidsequence of GKVAIILXGKHMGKRCIITK (SEQ ID NO: 40), wherein X is athreonine (T) residue or a serine (S) residue, (b) the amino acidsequence of GKHMGKRCIITKXLXSGLLAVVGPYE (SEQ ID NO: 41), wherein X is anisoleucine (I) residue, a valine (V) residue, an asparagine (N) residue,or a threonine (T) residue, (c) the amino acid sequence ofSGLLAVVGPYEXNGVPLKRV (SEQ ID NO: 42), wherein X is a valine (V) residueor an isoleucine (I) residue, and wherein each of the one or moreisolated polypeptides induces a protective immune response againstPlasmodium falciparum and/or Plasmodium vivax in a mammal.

The invention provides a composition comprising a pharmaceuticallyacceptable carrier and one or more isolated polypeptides, wherein eachof the one or more isolated polypeptides comprises (a) an amino acidsequence comprising at least 20 contiguous amino acid residues of thesequence LEKKMQLKKEGKLLSAKAKEEKKK (SEQ ID NO: 55), (b) an amino acidsequence comprising at least 21 contiguous amino acid residues of thesequence IVCILGHVDTGKTKLLDKLRHTNVQDNEAGGITQQIGATFFPKD (SEQ ID NO: 56),(c) an amino acid sequence comprising at least 21 contiguous amino acidresidues of the sequenceSKGIMIIDTPGHESFYNLRKRGSSLCDIAILVIDLMHGLEQQTKESIQILKQRNCPFVIALNKIDRLYMW(SEQ ID NO: 57), (d) an amino acid sequence comprising at least 20contiguous amino acid residues of the sequenceKLECTVLEVKNIEGLGTTIDVILTNG (SEQ ID NO: 58), (e) the amino acid sequenceof GVGLYVMASTLGSLEALLIFL (SEQ ID NO: 59), and (f) an amino acid sequencecomprising at least 20 contiguous amino acid residues of the sequenceMTDVSDVFNHVDKXGVGLYVMASTLGSLEALLIFL (SEQ ID NO: 60), wherein X is aserine (S) residue or a threonine (T) residue, and wherein each of theone or more isolated polypeptides induces a protective immune responseagainst Plasmodium falciparum and/or Plasmodium vivax in a mammal.

The invention provides a composition comprising a pharmaceuticallyacceptable carrier and one or more isolated polypeptides, wherein eachof the one or more isolated polypeptides comprises (a) the amino acidsequence of NLFFAKQVIPNACATQAILSI (SEQ ID NO: 69), or (b) the amino acidsequence of NFDSXMKGLTLSNCXFLRNIHN (SEQ ID NO: 70), wherein X is aserine (S) residue, a threonine (T) residue, or an asparagine (N)residue, and wherein each of the one or more isolated polypeptidesinduces a protective immune response against Plasmodium falciparumand/or Plasmodium vivax in a mammal.

The invention provides a composition comprising a pharmaceuticallyacceptable carrier and one or more isolated polypeptides, wherein eachof the one or more isolated polypeptides comprises (a) the amino acidsequence of SGWKFEEQEGDVNMVLTKNVD (SEQ ID NO: 80), (b) an amino acidsequence comprising at least 20 contiguous amino acid residues of thesequence IIDFQLVSPFQAEGENEAQAEMTDFSVTVEKPN (SEQ ID NO: 81), (c) an aminoacid sequence comprising at least 20 contiguous amino acid residues ofthe sequence GGITFYCTTLQNDEKFRYMIGNVKYYKNEEGKNSVS (SEQ ID NO: 82), and(d) an amino acid sequence comprising at least 21 contiguous amino acidresidues of the sequence YNGPEFEDLDDSLQTSLDEWLANLGVDSELCDFIDSCSIDKEQREYM(SEQ ID NO: 83), and wherein each of the one or more isolatedpolypeptides induces a protective immune response against Plasmodiumfalciparum and/or Plasmodium vivax in a mammal.

The invention provides a composition comprising a pharmaceuticallyacceptable carrier and one or more isolated polypeptides, wherein eachof the one or more isolated polypeptides comprises the amino acidsequence of SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93,or SEQ ID NO: 202, and wherein each of the one or more isolatedpolypeptides induces a protective immune response against Plasmodiumfalciparum and/or Plasmodium vivax in a mammal.

The invention provides a composition comprising a pharmaceuticallyacceptable carrier and one or more isolated polypeptides, wherein eachof the one or more isolated polypeptides comprises the amino acidsequence of SEQ ID NO: 98, SEQ ID NO: 99, or SEQ ID NO: 100, and whereineach of the one or more isolated polypeptides induces a protectiveimmune response against Plasmodium falciparum and/or Plasmodium vivax ina mammal.

The invention provides a composition comprising a pharmaceuticallyacceptable carrier and one or more isolated polypeptides, wherein eachof the one or more isolated polypeptides comprises (a) the amino acidsequence of LLKHGWCEMLKGGVIMDVKX (SEQ ID NO: 104), wherein X is anasparagine (N) residue or a serine (S) residue, (b) an amino acidsequence comprising at least 20 contiguous amino acid residues of thesequence VEQAKIAEXAGAIGVMVLENIPSELR (SEQ ID NO: 105), wherein X is alysine (K) residue or a glutamic acid (E) residue, (c) an amino acidsequence comprising at least 20 contiguous amino acid residues of thesequence SINVLAKVRIGHFVEAQILEELK (SEQ ID NO: 106), (d) an amino acidsequence comprising at least 27 contiguous amino acid residues of thesequence KHKFKTPFVCGCTNLGEALRRXSEGASMIRTKGEAGTGNII (SEQ ID NO: 107),wherein X is an isoleucine (I) residue or a methionine (M) residue, (e)an amino acid sequence comprising at least 20 contiguous amino acidresidues of the sequence ATPADAAMCMQLGMDGVFVGSGIFESENP (SEQ ID NO: 108),or (f) an amino acid sequence comprising at least 20 contiguous aminoacid residues of the sequence LPVVNFAAGGXATPADAAMCMQLGMDGVFVGSGIFESENP(SEQ ID NO: 109), wherein X is an isoleucine (I) residue or a valine (V)residue, and wherein each of the one or more isolated polypeptidesinduces a protective immune response against Plasmodium falciparumand/or Plasmodium vivax in a mammal.

The invention provides composition comprising a pharmaceuticallyacceptable carrier and one or more isolated nucleic acid sequences,wherein each of the one or more isolated nucleic acid sequences encodesa polypeptide comprising the amino acid sequence of any one of SEQ IDNO: 1-SEQ ID NO: 16, SEQ ID NO: 20-SEQ ID NO: 26, SEQ ID NO: 30-SEQ IDNO: 36, SEQ ID NO: 40-SEQ ID NO: 51, SEQ ID NO: 55-SEQ ID NO: 65, SEQ IDNO: 69-SEQ ID NO: 76, SEQ ID NO: 80-SEQ ID NO: 86, SEQ ID NO: 90-SEQ IDNO: 93, SEQ ID NO: 98-SEQ ID NO: 100, SEQ ID NO: 104-SEQ ID NO: 118, SEQID NO: 202, SEQ ID NO: 205, SEQ ID NO: 207-SEQ ID NO: 209, SEQ ID NO:211, or SEQ ID NO: 212, and wherein each of the one or more isolatedpolypeptides induces a protective immune response against Plasmodiumfalciparum and/or Plasmodium vivax in a mammal.

DETAILED DESCRIPTION OF THE INVENTION

The invention is predicated, at least in part, on the identification ofantigenic polypeptides from Plasmodium parasites that provide protectiveimmunity in mammals against challenge with malaria-causing parasites. Inthis respect, the invention provides a composition comprising apharmaceutically acceptable carrier and one or more isolatedpolypeptides or nucleic acid sequences, each of which comprises orencodes, respectively, a Plasmodium amino acid sequence, wherein thePlasmodium amino acid sequence induces a protective immune responseagainst Plasmodium falciparum and/or Plasmodium vivax in a mammal. Theone or more polypeptides are “isolated” in that they are removed fromtheir natural environment (i.e., a Plasmodium parasite).

Each of the one or more Plasmodium amino acid sequences is a Plasmodiumantigen. An “antigen” is a molecule that triggers an immune response ina mammal. An “immune response” can entail, for example, antibodyproduction and/or the activation of immune effector cells. An antigen inthe context of the invention can comprise any subunit, fragment, orepitope of any proteinaceous or non-proteinaceous (e.g., carbohydrate orlipid) molecule which provokes an immune response in a mammal. By“epitope” is meant a sequence of an antigen that is recognized by anantibody or an antigen receptor. Epitopes also are referred to in theart as “antigenic determinants.”

A Plasmodium antigen in the context of the invention can comprise anyproteinaceous Plasmodium molecule or portion thereof that provokes aPlasmodium-related immune response in a mammal. A “Plasmodium molecule”is a molecule that is a part of a Plasmodium parasite, is encoded by anucleic acid sequence of a Plasmodium parasite, or is derived from orsynthetically based upon any such molecule. Administration of aPlasmodium antigen that provokes an immune response in accordance withthe invention preferably leads to protective immunity againstPlasmodium. In this regard, an “immune response” to Plasmodium is animmune response to any one or more Plasmodium antigens.

The one or more Plasmodium amino acid sequences can be obtained orderived from any Plasmodium species. Preferably, the one or morePlasmodium amino acid sequences are derived or obtained from aPlasmodium species that infects humans and causes malaria.Human-infecting Plasmodium species include P. malariae, P. ovale, P.knowlesi, P. vivax, and P. falciparum. P. vivax and P. falciparum arethe most common species, while P. falciparum is the most deadly speciesof Plasmodium in human.

Alternatively, each of the one or more Plasmodium amino acid sequencescan be an ortholog of an amino acid sequence from a human-infectingPlasmodium species. “Orthologs” or “orthologous genes” are nucleic acidor amino acid sequences that evolved from a common ancestral gene byspeciation, and typically retain the same function in the course ofevolution. In other words, when a species diverges into two separatespecies, the copies of a single gene in the two resulting species aresaid to be “orthologous.” Plasmodium species that infect non-humananimals and contain orthologous genes include, for example,rodent-infecting species such as P. vinckei, P. chabaudi, P. yoelii, andP. berghei; and non-human primate-infecting species such as P. knowlesi,P. cynomolgi, P. simiovale, P. fieldi, P. inui, P. brasilianum, P.billbrayi, P. billcollinsi, P. bouillize, P. brasilianum, P. bucki, P.cercopitheci, P. coatneyi, P. coulangesi, P. eylesi, P. fieldi, P.foleyi, P. fragile, P. inui, P. gaboni, P. georgesi, P. girardi, and P.gonderi. In order to advance vaccine discovery, the genomes of a numberof Plasmodium species have been sequenced. For example, the complete P.falciparum genome has been sequenced and is disclosed in Gardner et al.,Nature, 419: 498-511 (2002). The complete P. vivax genome has beensequenced and is disclosed in Neafsey et al., Nature Genetics, 44:1046-1050 (2012); Carlton et al., Nature, 455: 757-763 (2008); andDharia et al., Proc. Natl. Acad. Sci. USA, 107: 20045-20050 (2010). Inaddition, the complete P. yoelii genome sequence is disclosed in Carltonet al., Nature, 419: 512-9 (2002). Mouse models of malaria infection, inwhich P. yoelii is the infecting parasite, have been generated (see,e.g., Wiersch et al., Methods in Molecular Medicine, 72: Malaria Methodsand Protocols: 57-76 (2002); and Voza et al., Malaria Journal, 9: 362(2010)), and have been established as reliable models forpre-erythrocytic stage P. falciparum vaccine development (see, e.g.,Nussenzweig et al., Nature, 216: 160 (1967); Clyde et al., Am. J. Med.Sci., 266: 169 (1973); Roeckmann et al., Trans R. Soc. Trop. Med. Hyg.,68: 258 (1974); Hoffman et al., J. Inf. Dis., 185: 1155 (2002);Schofield et al., Nature, 330: 64 (1987); Weiss et al., Proc. Natl.Acad. Sci. USA, 85: 573 (1988); Kumar et al., Nature, 444: 937 (2006);Stoute et al., N. Eng. J. Med., 336: 86 (1997)).

In one embodiment, the composition comprises a pharmaceuticallyacceptable carrier and one or more isolated polypeptides, wherein eachof the one or more isolated polypeptides comprises: (a) the amino acidsequence of KKERYXIWXRXPKXELHCHLD (SEQ ID NO: 1), wherein X is a lysine(K) residue, a glutamic acid (E) residue, an arginine (R) residue, anisoleucine (I) residue, a leucine (L) residue, a cysteine (C) residue,or a valine (V) residue, (b) the amino acid sequence of KYKEGVVLMEFRYSP(SEQ ID NO: 2), (c) an amino acid sequence comprising at least 20contiguous amino acid residues of the sequence EDLAKXAVXXKYKEGVVLMEFRYSP(SEQ ID NO: 3), wherein X is a histidine (H) residue, a tryptophan (W)residue, a phenylalanine (F) residue, an isoleucine (I) residue, anasparagine (N) residue, or a glutamic acid (E) residue, or (d) an aminoacid sequence comprising at least 20 contiguous amino acid residues ofthe sequence KSMDTHPIRXLYDAGVKVSVNSDDPGMFL (SEQ ID NO: 4), wherein X isa glutamine (Q) residue, a methionine (M) residue, or a lysine (K)residue.

When the composition comprises an isolated polypeptide comprising theamino acid sequence of SEQ ID NO: 1, X can be any suitable amino acidresidue, but preferably is a lysine (K) residue, a glutamic acid (E)residue, an arginine (R) residue, an isoleucine (I) residue, a leucine(L) residue, a cysteine (C) residue, or a valine (V) residue. In thisrespect, the isolated polypeptide can comprise, for example, the aminoacid sequence of KKERYEIWRRIPKVELHCHLD (SEQ ID NO: 5),KKERYKIWKRLPKCELHCHLD (SEQ ID NO: 6), or KKERYKIWKRIPKCELHCHLD (SEQ IDNO: 7). When the composition comprises an amino acid sequence comprisingat least 20 contiguous amino acid residues (e.g., 20, 21, 22, 23, 24, or25 contiguous amino acid residues) of the sequence SEQ ID NO: 3, X canbe any suitable amino acid residue, but preferably is a histidine (H)residue, a tryptophan (W) residue, a phenylalanine (F) residue, anisoleucine (I) residue, an asparagine (N) residue, or a glutamic acid(E) residue. In this respect, the isolated polypeptide can comprise, forexample, an amino acid sequence comprising at least 20 contiguous aminoacid residues of the sequence EDLAKWAVIEKYKEGVVLMEFRYSP (SEQ ID NO: 8),EDLAKHAVFNKYKEGVVLMEFRYSP (SEQ ID NO: 9), or EDLAKHAVFNKYKEGVVLMEFRYSP(SEQ ID NO: 10). In particular, the isolated polypeptide can comprisethe amino acid sequence of EDLAKWAVIEKYKEGVVLMEFRYSP (SEQ ID NO: 8),EDLAKHAVFNKYKEGVVLMEFRYSP (SEQ ID NO: 9), or EDLAKHAVFNKYKEGVVLMEFRYSP(SEQ ID NO: 10). When the composition comprises an isolated polypeptidecomprising at least 20 contiguous amino acid residues (e.g., 20, 21, 22,23, 24, 25, 26, 27, 28, or 29 contiguous amino acid residues) of SEQ IDNO: 4, the isolated polypeptide can comprise, for example, at least 20contiguous amino acid residues of the sequenceKSMDTHPIRKLYDAGVKVSVNSDDPGMFL (SEQ ID NO: 11),KSMDTHPIRMLYDAGVKVSVNSDDPGMFL (SEQ ID NO: 12), orKSMDTHPIRQLYDAGVKVSVNSDDPGMFL (SEQ ID NO: 13). In particular, theisolated polypeptide can comprise the amino acid sequence ofKSMDTHPIRKLYDAGVKVSVNSDDPGMFL (SEQ ID NO: 11),KSMDTHPIRMLYDAGVKVSVNSDDPGMFL (SEQ ID NO: 12), orKSMDTHPIRQLYDAGVKVSVNSDDPGMFL (SEQ ID NO: 13).

The composition can comprise one, two, three, or all four, of theaforementioned polypeptides alone or in any combination. In thisrespect, the composition can comprise any combination of any two of theaforementioned sequences, any combination of any three of theaforementioned sequences, or all four of the aforementioned sequences.In a preferred embodiment, the composition comprises an isolatedpolypeptide comprising the amino acid sequence of SEQ ID NO: 14, SEQ IDNO: 15, SEQ ID NO: 16, or SEQ ID NO: 209.

In another embodiment, the composition comprises a pharmaceuticallyacceptable carrier and one or more isolated polypeptides, wherein eachof the one or more isolated polypeptides comprises (a) an amino acidsequence comprising at least 20 contiguous amino acid residues (e.g.,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 contiguousamino acid residues) of the sequence MKKDREPIDEDEMRITSTGRMTNYVNYGAKILG(SEQ ID NO: 20), (b) an amino acid sequence comprising at least 20contiguous amino acid residues (e.g., 20, 21, 22, 23, 24, 25, 26, 27,28, 29, or 30 contiguous amino acid residues) of the sequenceKIKATGNAIGKAVTLAEIIKRRFKGLHQIT (SEQ ID NO: 21), or (c) the amino acidsequence of SEQ ID NO: 22.

When the composition comprises an isolated polypeptide comprising anamino acid sequence comprising at least 20 contiguous amino acidresidues of SEQ ID NO: 20, the isolated polypeptide can comprise, forexample, the amino acid sequence of SEQ ID NO: 20. When the compositioncomprises an isolated polypeptide comprising an amino acid sequencecomprising at least 20 contiguous amino acid residues of SEQ ID NO: 21,the isolated polypeptide can comprise, for example, the amino acidsequence of SEQ ID NO: 21. When the composition comprises an isolatedpolypeptide comprising the amino acid sequence of SEQ ID NO: 22, theisolated polypeptide can comprise, for example, the amino acid sequenceof KDAGYQPPLDEKYVKEMXPEEIVN (SEQ ID NO: 23), wherein X can be anysuitable amino acid residue, but is preferably a serine (S) residue or athreonine (T) residue (i.e., KDAGYQPPLDEKYVKEMXPEEIVN (SEQ ID NO: 211)or KDAGYQPPLDEKYVKEMTPEEIVN (SEQ ID NO: 212)).

The composition can comprise one, two, or all three of theaforementioned polypeptides alone or in any combination. In thisrespect, the composition can comprise any combination of any two of theaforementioned sequences, or all three of the aforementioned sequences.In a preferred embodiment, the composition comprises an isolatedpolypeptide comprising the amino acid sequence of SEQ ID NO: 24, SEQ IDNO: 25, or SEQ ID NO: 26.

In another embodiment, the composition comprises a pharmaceuticallyacceptable carrier and one or more isolated polypeptides, wherein eachof the one or more isolated polypeptides comprises (a) an amino acidsequence which comprises at least 40 contiguous amino acid residues ofthe sequenceMGKEKTHINLVVIGHVDSGKSTTTGHIIYKLGGIDRRTIEKFEKESAEMGKGSFKYAWVLDKLKAERERGITIDIALWKFETPRYFFTVIDAPGHKDFIKNMITGTSQADVALLVVPAEVGGFEGAFSKEGQTKEHALLAFTLGVKQIVVGVNKMDTVKYSEDRYEEIKKEV(SEQ ID NO: 30), (b) an amino acid sequence which comprises at least 55contiguous amino acid residues of the sequenceDYLKKVGYQADKVDFIPISGFEGDNLIEKSDKTPWYKGRTLIEALDTMEPPKRPYDKPLRIPLQGVYKIGGIGTVPVGRVETGILKAGMVLNFAPSAVVSECKSVEMHKEVLEEARPGDNIGFNVKNVSVKEIKRGYVASDTKNEPAKGCSKFTAQVIILNHPGEIKNGY(SEQ ID NO: 31), (c) the amino acid sequence ofHISCKFLNIDSKIDKRSGKVVEENPK (SEQ ID NO: 32), or (d) an amino acidsequence which comprises at least 20 contiguous amino acid residues ofthe sequence LEPKKPMVVETFTEYPPLGRFAIRDMRQTIAVGIIK (SEQ ID NO: 33).

When the composition comprises an isolated polypeptide comprising atleast 40 contiguous amino acid residues (e.g., 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140,145, 150 155, 160, 165, 170, or 172 contiguous amino acid residues) ofSEQ ID NO: 30, the isolated polypeptide can comprise, for example, SEQID NO: 30. When the composition comprises an isolated polypeptidecomprising at least 55 contiguous amino acid residues (e.g., 55, 60, 65,70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140,145, 150 155, 160, 165, or 170 contiguous amino acid residues) of SEQ IDNO: 31, the isolated polypeptide can comprise, for example, SEQ ID NO:31. When the composition comprises an isolated polypeptide comprising atleast 20 contiguous amino acid residues (e.g., 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32 or 33 contiguous amino acid residues) of SEQID NO: 33, the isolated polypeptide can comprise, for example, SEQ IDNO: 33.

The composition can comprise one, two, three or all four of theaforementioned polypeptides alone or in any combination. In thisrespect, the composition can comprise any combination of any two of theaforementioned sequences, any combination of any three of theaforementioned sequences, or all four of the aforementioned sequences.In a preferred embodiment, the composition comprises an isolatedpolypeptide comprising the amino acid sequence of SEQ ID NO: 34, SEQ IDNO: 35, or SEQ ID NO: 36.

In another embodiment, the composition comprises a pharmaceuticallyacceptable carrier and one or more isolated polypeptides, wherein eachof the one or more isolated polypeptides comprises (a) the amino acidsequence of GKVAIILXGKHMGKRCIITK (SEQ ID NO: 40), wherein X is athreonine (T) residue or a serine (S) residue, (b) the amino acidsequence of GKHMGKRCIITKXLXSGLLAVVGPYE (SEQ ID NO: 41), wherein X is anisoleucine (I) residue, a valine (V) residue, an asparagine (N) residue,or a threonine (T) residue, (c) the amino acid sequence ofSGLLAVVGPYEXNGVPLKRV (SEQ ID NO: 42), wherein X is a valine (V) residueor an isoleucine (I) residue.

When the composition comprises an isolated polypeptide comprising theamino acid sequence of GKVAIILXGKHMGKRCIITK (SEQ ID NO: 40), X can beany suitable amino acid residue, but preferably is a threonine (T)residue or a serine (S) residue. In this respect, the isolatedpolypeptide can comprise, for example, the amino acid sequence ofGKVAIILTGKHMGKRCIITK (SEQ ID NO: 43) or GKVAIILSGKHMGKRCIITK (SEQ ID NO:44). When the composition comprises an isolated polypeptide comprisingthe amino acid sequence of GKHMGKRCIITKXLXSGLLAVVGPYE (SEQ ID NO: 41), Xcan be any suitable amino acid residue, but preferably is an isoleucine(I) residue, a valine (V) residue, an asparagine (N) residue, or athreonine (T) residue. In this respect, the isolated polypeptide cancomprise, for example, the amino acid sequence ofGKHMGKRCIITKILNSGLLAVVGPYE (SEQ ID NO: 45) or GKHMGKRCIITKVLTSGLLAVVGPYE(SEQ ID NO: 46). When the composition comprises an isolated polypeptidecomprising the amino acid sequence of SGLLAVVGPYEXNGVPLKRV (SEQ ID NO:42), X can be any suitable amino acid residue, but preferably is avaline (V) residue or an isoleucine (I) residue. In this respect, theisolated polypeptide can comprise, for example, the amino acid sequenceof SGLLAVVGPYEVNGVPLKRV (SEQ ID NO: 47) or SGLLAVVGPYEINGVPLKRV (SEQ IDNO: 48).

The composition can comprise one, two, or all three of theaforementioned polypeptides alone or in any combination. In thisrespect, the composition can comprise any combination of any two of theaforementioned sequences, or all three of the aforementioned sequences.In a preferred embodiment, the composition comprises an isolatedpolypeptide comprising the amino acid sequence of SEQ ID NO: 49, SEQ IDNO: 50, or SEQ ID NO: 51.

In another embodiment, the composition comprises a pharmaceuticallyacceptable carrier and one or more isolated polypeptides, wherein eachof the one or more isolated polypeptides comprises (a) an amino acidsequence comprising at least 20 contiguous amino acid residues of thesequence LEKKMQLKKEGKLLSAKAKEEKKK (SEQ ID NO: 55), (b) an amino acidsequence comprising at least 21 contiguous amino acid residues of thesequence IVCILGHVDTGKTKLLDKLRHTNVQDNEAGGITQQIGATFFPKD (SEQ ID NO: 56),(c) an amino acid sequence comprising at least 21 contiguous amino acidresidues of the sequence the amino acid sequence ofSKGIMIIDTPGHESFYNLRKRGSSLCDIAILVIDLMHGLEQQTKESIQILKQRNCPFVIALNKIDRLYMW(SEQ ID NO: 57), (d) an amino acid sequence comprising at least 20contiguous amino acid residues of the sequenceKLECTVLEVKNIEGLGTTIDVILTNG (SEQ ID NO: 58), (e) the amino acid sequenceof GVGLYVMASTLGSLEALLIFL (SEQ ID NO: 59), and (f) an amino acid sequencecomprising at least 20 contiguous amino acid residues of the sequenceMTDVSDVFNHVDKXGVGLYVMASTLGSLEALLIFL (SEQ ID NO: 60), wherein X is aserine (S) residue or a threonine (T) residue.

When the composition comprises an isolated polypeptide comprising atleast 20 contiguous amino acid residues (e.g., 20, 21, 22, 23, or 24contiguous amino acid residues) of SEQ ID NO: 55, the isolatedpolypeptide can comprise, for example, SEQ ID NO: 55. When thecomposition comprises an isolated polypeptide comprising an amino acidsequence comprising at least 21 contiguous amino acid residues (22, 23,24 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, or 44 contiguous amino acid residues) of SEQ ID NO: 56, theisolated polypeptide can comprise, for example, SEQ ID NO: 56. When thecomposition comprises an isolated polypeptide comprising at least 21contiguous amino acid residues (e.g., 21, 25, 30, 35, 40, 45, 50, 55,60, 65, or 70 contiguous amino acid residues) of SEQ ID NO: 57, theisolated polypeptide can comprise, for example, SEQ ID NO: 57. When thecomposition comprises an isolated polypeptide comprising at least 20contiguous amino acid residues (e.g., 20, 21, 22, 23, 24, 25, or 26contiguous amino acid sequences) of SEQ ID NO: 58, the isolatedpolypeptide can comprise, for example, SEQ ID NO: 58. When thecomposition comprises an isolated polypeptide comprising at least 20contiguous amino acid residues (e.g., 20, 21, 22, 23, 24 25, 26, 27, 28,29, 30, 31, 32, 33, 34, or 35 contiguous amino acid residues) of SEQ IDNO: 60, X can be any suitable amino acid residue, but preferably is aserine (S) residue or a threonine (T) residue. In this respect, theisolated polypeptide can comprise, for example, an isolated polypeptidecomprising at least 20 contiguous amino acid residues of the sequenceMTDVSDVFNHVDKSGVGLYVMASTLGSLEALLIFL (SEQ ID NO: 61) orMTDVSDVFNHVDKTGVGLYVMASTLGSLEALLIFL (SEQ ID NO: 62). In particular, theisolated polypeptide can comprise, for example, the amino acid sequenceof MTDVSDVFNHVDKSGVGLYVMASTLGSLEALLIFL (SEQ ID NO: 61) orMTDVSDVFNHVDKTGVGLYVMASTLGSLEALLIFL (SEQ ID NO: 62).

The composition can comprise one, two, three, four, five, or all six ofthe aforementioned polypeptides alone or in any combination. In thisrespect, the composition can comprise any combination of any two of theaforementioned sequences, any combination of any three of theaforementioned sequences, any combination of any four of theaforementioned sequences, any combination of any five of theaforementioned sequences, or all six of the aforementioned sequences. Ina preferred embodiment, the composition comprises an isolatedpolypeptide comprising the amino acid sequence of SEQ ID NO: 63, SEQ IDNO: 64, or SEQ ID NO: 65.

In another embodiment, the composition comprises a pharmaceuticallyacceptable carrier and one or more isolated polypeptides, wherein eachof the one or more isolated polypeptides comprises (a) the amino acidsequence of NLFFAKQVIPNACATQAILSI (SEQ ID NO: 69), and (b) the aminoacid sequence of NFDSXMKGLTLSNCXFLRNIHN (SEQ ID NO: 70), wherein X is aserine (S) residue, a threonine (T) residue, or an asparagine (N)residue.

When the composition comprises the amino acid sequence of SEQ ID NO: 70,X can be any suitable amino acid residue, but preferably is a serine (S)residue, a threonine (T) residue, or an asparagine (N) residue. In thisrespect, the isolated polypeptide can comprise, for example, the aminoacid sequence of NFDSTMKGLTLSNCNFLRNIHN (SEQ ID NO: 71),NFDSSMKGLTLSNCTFLRNIHN (SEQ ID NO: 72), or NFDSSMKGLTLSNCNFLRNIHN (SEQID NO: 73). The composition can comprise one or both of theaforementioned polypeptides. In a preferred embodiment, the compositioncomprises an isolated polypeptide comprising the amino acid sequence ofSEQ ID NO: 74, SEQ ID NO: 75, or SEQ ID NO: 76.

In another embodiment, the composition comprises a pharmaceuticallyacceptable carrier and one or more isolated polypeptides, wherein eachof the one or more isolated polypeptides comprises (a) the amino acidsequence of SGWKFEEQEGDVNMVLTKNVD (SEQ ID NO: 80), (b) an amino acidsequence comprising at least 20 contiguous amino acid residues of thesequence IIDFQLVSPFQAEGENEAQAEMTDFSVTVEKPN (SEQ ID NO: 81), (c) an aminoacid sequence comprising at least 20 contiguous amino acid residues ofthe sequence GGITFYCTTLQNDEKFRYMIGNVKYYKNEEGKNSVS (SEQ ID NO: 82), and(d) an amino acid sequence comprising at least 21 contiguous amino acidresidues of the sequence YNGPEFEDLDDSLQTSLDEWLANLGVDSELCDFIDSCSIDKEQREYM(SEQ ID NO: 83).

When the composition comprises an isolated polypeptide comprising atleast 20 contiguous amino acid residues (e.g., 20, 21, 22, 23, 24 25,26, 27, 28, 29, 30, 31, 32, or 33 contiguous amino acid residues) of SEQID NO: 81, the isolated polypeptide can comprise, for example, SEQ IDNO: 81. When the composition comprises an isolated polypeptidecomprising at least 20 contiguous amino acid residues (e.g., 20, 21, 22,23, 24 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 contiguous aminoacid residues) of SEQ ID NO: 82, the isolated polypeptide can comprise,for example, SEQ ID NO: 82. When the composition comprises an isolatedpolypeptide comprising at least 21 contiguous amino acid residues of SEQID NO: 83, (e.g., 21, 22, 23, 24 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, or 47 contiguous aminoacid residues), the isolated polypeptide can comprise, for example, SEQID NO: 83.

The composition can comprise one, two, or all three of theaforementioned polypeptides alone or in any combination. In thisrespect, the composition can comprise any combination of any two of theaforementioned sequences, or all three of the aforementioned sequences.In a preferred embodiment, the composition comprises an isolatedpolypeptide comprising the amino acid sequence of SEQ ID NO: 84, SEQ IDNO: 85, or SEQ ID NO: 86.

In another embodiment, the composition comprises a pharmaceuticallyacceptable carrier and one or more isolated polypeptides, each of whichcomprises the amino acid sequence of SEQ ID NO: 90, SEQ ID NO: 91, SEQID NO: 92, SEQ ID NO: 93, or SEQ ID NO: 202. The invention also providesa composition comprising a pharmaceutically acceptable carrier and oneor more isolated polypeptides, each of which comprises the amino acidsequence of SEQ ID NO: 98, SEQ ID NO: 99, or SEQ ID NO: 100.

In another embodiment, the composition comprises a pharmaceuticallyacceptable carrier and one or more isolated polypeptides, wherein eachof the one or more isolated polypeptides comprises (a) the amino acidsequence of LLKHGWCEMLKGGVIMDVKX (SEQ ID NO: 104), wherein X is anasparagine (N) residue or a serine (S) residue, (b) an amino acidsequence comprising at least 20 contiguous amino acid residues of thesequence VEQAKIAEXAGAIGVMVLENIPSELR (SEQ ID NO: 105), wherein X is alysine (K) residue or a glutamic acid (E) residue, (c) an amino acidsequence comprising at least 20 contiguous amino acid residues of thesequence SINVLAKVRIGHFVEAQILEELK (SEQ ID NO: 106), (d) an amino acidsequence comprising at least 27 contiguous amino acid residues of thesequence KHKFKTPFVCGCTNLGEALRRXSEGASMIRTKGEAGTGNII (SEQ ID NO: 107),wherein X is an isoleucine (I) residue or a methionine (M) residue, (e)an amino acid sequence comprising at least 20 contiguous amino acidresidues of the sequence ATPADAAMCMQLGMDGVFVGSGIFESENP (SEQ ID NO: 108),or (f) an amino acid sequence comprising at least 20 contiguous aminoacid residues of the sequence LPVVNFAAGGXATPADAAMCMQLGMDGVFVGSGIFESENP(SEQ ID NO: 109), wherein X is an isoleucine (I) residue or a valine (V)residue.

When the composition comprises an isolated polypeptide comprising theamino acid sequence of LLKHGWCEMLKGGVIMDVKX (SEQ ID NO: 104), X can beany suitable amino acid residue, but preferably is an asparagine (N)residue or a serine (S) residue. In this respect, the isolatedpolypeptide can comprise, for example, the amino acid sequence ofLLKHGWCEMLKGGVIMDVKN (SEQ ID NO: 110), or LLKHGWCEMLKGGVIMDVKS (SEQ IDNO: 111). When the composition comprises an isolated polypeptidecomprising at least 20 contiguous amino acid residues (e.g., 20, 21, 22,23, 24, 25, or 26 contiguous amino acid residues) of SEQ ID NO: 105, Xcan be any suitable amino acid residue, but preferably is a lysine (K)residue or a glutamic acid (E) residue. In this respect, the isolatedpolypeptide can comprise, for example, at least 20 contiguous amino acidresidues of the sequence VEQAKIAEKAGAIGVMVLENIPSELR (SEQ ID NO: 112), orVEQAKIAEEAGAIGVMVLENIPSELR (SEQ ID NO: 113). In particular, the isolatedpolypeptide can comprise, for example, the amino acid sequence ofVEQAKIAEKAGAIGVMVLENIPSELR (SEQ ID NO: 112), orVEQAKIAEEAGAIGVMVLENIPSELR (SEQ ID NO: 113). When the compositioncomprises an isolated polypeptide comprising at least 20 contiguousamino acid residues (e.g., 20, 21, 22, or 23 contiguous amino acidresidues) of SEQ ID NO: 106, the isolated polypeptide can comprise, forexample, SEQ ID NO: 106. When the composition comprises an isolatedpolypeptide comprising at least 27 contiguous amino acid residues (e.g.,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or 41 contiguousamino acid residues) of SEQ ID NO: 107, X can be any suitable amino acidresidue, but preferably is an isoleucine (I) residue or a methionine (M)residue. In this respect, the isolated polypeptide can comprise, forexample, at least 27 contiguous amino acid residues ofKHKFKTPFVCGCTNLGEALRRISEGASMIRTKGEAGTGNII (SEQ ID NO: 207), orKHKFKTPFVCGCTNLGEALRRMSEGASMIRTKGEAGTGNII (SEQ ID NO: 208). Inparticular, the isolated polypeptide can comprise, for example, theamino acid sequence of KHKFKTPFVCGCTNLGEALRRISEGASMIRTKGEAGTGNII (SEQ IDNO: 207), or KHKFKTPFVCGCTNLGEALRRMSEGASMIRTKGEAGTGNII (SEQ ID NO: 208).When the composition comprises an isolated polypeptide comprising atleast 20 contiguous amino acid residues (e.g., 20, 21, 22, 23, 24, 25,26, 27, 28, or 29 contiguous amino acid residues) of SEQ ID NO: 108, theisolated polypeptide can comprise, for example SEQ ID NO: 108. When thecomposition comprises an isolated polypeptide comprising least 20contiguous amino acid residues (e.g., 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 contiguous aminoacid residues) of SEQ ID NO: 109, X can be any suitable amino acidresidue, but preferably is an isoleucine (I) residue or a valine (V)residue. In this respect, the isolated polypeptide can comprise, forexample, at least 20 contiguous amino acid residues ofLPVVNFAAGGIATPADAAMCMQLGMDGVFVGSGIFESENP (SEQ ID NO: 114), orLPVVNFAAGGVATPADAAMCMQLGMDGVFVGSGIFESENP (SEQ ID NO: 115). Inparticular, the isolated polypeptide can comprise, for example,LPVVNFAAGGIATPADAAMCMQLGMDGVFVGSGIFESENP (SEQ ID NO: 114), orLPVVNFAAGGVATPADAAMCMQLGMDGVFVGSGIFESENP (SEQ ID NO: 115).

The composition can comprise one, two, three, four, five, or all six ofthe aforementioned polypeptides alone or in any combination. In thisrespect, the composition can comprise any combination of any two of theaforementioned sequences, any three of the aforementioned sequences, anyfour of the aforementioned sequences, any five of the aforementionedsequences, or all six of the aforementioned sequences. In a preferredembodiment, the composition comprises an isolated polypeptide comprisingthe amino acid sequence of SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO:118, or SEQ ID NO: 205.

The polypeptides of the inventive composition can be prepared by anymethod, such as by synthesizing the polypeptide or by expressing anucleic acid encoding an appropriate amino acid sequence in a cell andharvesting the peptide from the cell or media containing the cell. Acombination of such methods also can be used. Methods of de novosynthesizing polypeptides and methods of recombinantly producingpolypeptides are known in the art (see, e.g., Chan et al., Fmoc SolidPhase Peptide Synthesis, Oxford University Press, Oxford, United Kingdom(2005); Reid, R. (ed.), Peptide and Protein Drug Analysis, MarcelDekker, Inc. (2000); Westwood et al. (ed.), Epitope Mapping, OxfordUniversity Press, Oxford, United Kingdom (2000); Sambrook et al.,Molecular Cloning: A Laboratory Manual, 3^(rd) ed., Cold Spring HarborPress, Cold Spring Harbor, N.Y. (2001); and Ausubel et al., CurrentProtocols in Molecular Biology, Greene Publishing Associates and JohnWiley & Sons, New York (1994)).

In another embodiment of the invention, the composition can comprise apharmaceutically acceptable carrier and one or more isolated nucleicacid sequences, each of which encodes any of the aforementioned isolatedpolypeptides. In this respect, the composition can comprise one or moreisolated nucleic acid sequences, wherein each of the one or more nucleicacid sequences encodes a polypeptide comprising the amino acid sequenceof any one of SEQ ID NO: 1-SEQ ID NO: 16, SEQ ID NO: 20-SEQ ID NO: 26,SEQ ID NO: 30-SEQ ID NO: 36, SEQ ID NO: 40-SEQ ID NO: 51, SEQ ID NO:55-SEQ ID NO: 65, SEQ ID NO: 69-SEQ ID NO: 76, SEQ ID NO: 80-SEQ ID NO:86, SEQ ID NO: 90-SEQ ID NO: 93, SEQ ID NO: 98-SEQ ID NO: 100, SEQ IDNO: 104-SEQ ID NO: 118, SEQ ID NO: 202, SEQ ID NO: 205, SEQ ID NO:207-SEQ ID NO: 209, SEQ ID NO: 211, or SEQ ID NO: 212.

In one embodiment, the one or more isolated nucleic acid sequencescomprise codons expressed more frequently (and preferably, mostfrequently) in humans than in Plasmodium. While the genetic code isgenerally universal across species, the choice among synonymous codonsis often species-dependent. One of ordinary skill in the art wouldappreciate that, to achieve maximum protection against Plasmodiuminfection, high levels of Plasmodium antigens must be expressed in amammalian, preferably a human, host. In this respect, the nucleic acidsequence preferably encodes the native amino acid sequence of aPlasmodium antigen, but comprises codons that are expressed morefrequently in mammals (e.g., humans) than in Plasmodium. Changing nativePlasmodium codons to the most frequently used in mammals will increaseexpression of the Plasmodium antigen in a mammal (e.g., a human). Suchmodified nucleic acid sequences are commonly described in the art as“humanized,” as “codon-optimized,” or as utilizing “mammalian-preferred”or “human-preferred” codons

In the context of the invention, a Plasmodium nucleic acid sequence issaid to be “codon-optimized” if at least about 60% (e.g., at least about70%, at least about 80%, or at least about 90%) of the wild-type codonsin the nucleic acid sequence are encoded by mammalian-preferred codons.That is, a Plasmodium nucleic acid sequence is codon-optimized if atleast about 60% of the codons in the nucleic acid sequence aremammalian-preferred codons.

Alternatively, the one or more isolated nucleic acid sequences canutilize particular codons at frequencies that best approximate codonusage frequencies of native Plasmodium species. Methods and algorithmshave been developed in order to facilitate and adjust codon usage inthis manner when expressing genes in heterologous systems, and thesemethods are referred to as “codon harmonization” (see, e.g., U.S. PatentApplication Publication 2004/0209323; Angov et al., PLoS ONE, 3(5):e2189 (2008); and Angov et al., Methods Mol. Biol., 705: 1-13 (2011)).Adjusting codons of the isolated nucleic acid sequence so as to mimicthe codon usage of a native Plasmodium gene may improve gene and/orprotein expression by, for example, improving protein folding or proteinsolubility. Such modified nucleic acid sequences are commonly describedin the art as “harmonized” or “codon-harmonized.”

Additionally and alternatively, the codon-optimized or codon-harmonizednucleic acid sequence encoding a Plasmodium antigen can be any sequencethat hybridizes to an above-described sequence under at least moderate,preferably high, stringency conditions, such as described in, e.g.,Sambrook et al., Molecular Cloning, a Laboratory Manual, 3rd edition,Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (2001). Determiningthe degree of homology can be accomplished using any suitable methodknown in the art, such as those described herein.

In a preferred embodiment, the composition can comprise any one, orcombination of, the following nucleic acid sequences: SEQ ID NO: 17, SEQID NO: 18, SEQ ID NO: 19, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29,SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 52, SEQ ID NO:53, SEQ ID NO: 54, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ IDNO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 87, SEQ ID NO: 88, SEQID NO: 89, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97,SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 119, SEQ IDNO: 120, SEQ ID NO: 121, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 206,or SEQ ID NO: 210.

In one embodiment of the invention, the one or more isolated nucleicacid sequences which encode the Plasmodium antigens are present in avector. Any vector can be employed in the context of the invention,including viral and non-viral vectors. Examples of suitable viralvectors include, but are not limited to, retroviral vectors,adeno-associated virus vectors, poxviral vectors (e.g., vaccinia virusvectors), herpesvirus vectors, parainfluenza-RSV chimeric vectors(PIV-RSV), adenoviral vectors, poliovirus, alphavirus, baculovirus, andSindbis virus. Examples of suitable non-viral vectors include, but arenot limited to, plasmids (e.g., DNA plasmids), yeast (e.g.,Saccharomyces), liposomes, nanoparticles, and molecular conjugates(e.g., transferrin). When the vector is a plasmid (e.g., DNA plasmid),the plasmid can be administered with adjuvants, such as CpG or polymericadjuvants. The vector also can be a virus-like particle (VLP) (see,e.g., Petry et al., Curr. Opin. Molecular Therapeutics, 5(5): 524-528(2003); and U.S. Pat. No. 5,298,244).

In a preferred embodiment, the vector is an adenoviral vector. The term“adenoviral vector,” as used herein, refers to an adenovirus in whichthe adenoviral genome has been manipulated to accommodate one or morenucleic acid sequences that are non-native with respect to theadenoviral genome. Typically, an adenoviral vector is generated byintroducing one or more mutations (e.g., a deletion, insertion, orsubstitution) into the adenoviral genome of the adenovirus so as toaccommodate the insertion of a non-native nucleic acid sequence, forexample, for gene transfer, into the adenovirus.

Non-human adenovirus (e.g., ape, simian, avian, canine, ovine, or bovineadenoviruses) can be used to generate the adenoviral vector (i.e., as asource of the adenoviral genome for the adenoviral vector). For example,the adenoviral vector can be based on a simian adenovirus, includingboth new world and old world monkeys (see, e.g., Virus Taxonomy: VIIIthReport of the International Committee on Taxonomy of Viruses (2005)). Aphylogeny analysis of adenoviruses that infect primates is disclosed in,e.g., Roy et al., PLoS Pathog., 5(7): e100050.doi:10.1371/journal.ppat.1000503 (2009). For instance, a simianadenovirus can be of genotype 1, 3, 6, 7, 11, 16, 18, 19, 20, 27, 33,38, 39, 48, 49, or 50, or any other simian adenoviral genotype. Othernon-human adenoviruses which can be used in the invention includenon-human primate adenoviruses that are genetically and/orphenotypically similar to or distinct from group C human adenoviruses.

A gorilla adenovirus can be used as the source of the adenoviral genomefor the adenoviral vector. There are four widely recognized gorillasubspecies within the two species of Eastern Gorilla (Gorilla beringei)and Western Gorilla (Gorilla gorilla). The Western Gorilla speciesincludes the subspecies Western Lowland Gorilla (Gorilla gorillagorilla) and Cross River Gorilla (Gorilla gorilla diehli). The EasternGorilla species includes the subspecies Mountain Gorilla (Gorillaberingei beringei) and Eastern Lowland Gorilla (Gorilla beringeigraueri) (see, e.g., Wilson and Reeder, eds., Mammalian Species of theWorld, 3rd ed., Johns Hopkins University Press, Baltimore, Md. (2005)).The adenoviral vector can be based on an adenovirus isolated from any ofthe aforementioned subspecies. Preferably, the adenoviral vector isbased on an adenovirus isolated from Mountain Gorilla (Gorilla beringeiberingei) or Eastern Lowland Gorilla (Gorilla beringei graueri). Gorillaadenoviruses and adenoviral vectors are described in, e.g.,International Patent Application Nos. PCT/US2012/058956,PCT/US2012/058978, and PCT/US2012/059006.

A human adenovirus can be used as the source of the adenoviral genomefor the adenoviral vector. For instance, an adenovirus can be ofsubgroup A (e.g., serotypes 12, 18, and 31), subgroup B (e.g., serotypes3, 7, 11, 14, 16, 21, 34, 35, and 50), subgroup C (e.g., serotypes 1, 2,5, and 6), subgroup D (e.g., serotypes 8, 9, 10, 13, 15, 17, 19, 20,22-30, 32, 33, 36-39, and 42-48), subgroup E (e.g., serotype 4),subgroup F (e.g., serotypes 40 and 41), an unclassified serogroup (e.g.,serotypes 49 and 51), or any other adenoviral serogroup or serotype.Adenoviral serotypes 1 through 51 (i.e., Ad1 through Ad51) are availablefrom the American Type Culture Collection (ATCC, Manassas, Va.).Non-group C adenoviral vectors, methods of producing non-group Cadenoviral vectors, and methods of using non-group C adenoviral vectorsare disclosed in, for example, U.S. Pat. Nos. 5,801,030, 5,837,511, and5,849,561, and International Patent Application Publications WO1997/012986 and WO 1998/053087.

The adenoviral vector can comprise a combination of subtypes and therebybe a “chimeric” adenoviral vector. A chimeric adenoviral vector cancomprise an adenoviral genome that is derived from two or more (e.g., 2,3, 4, etc.) different adenovirus serotypes. In the context of theinvention, a chimeric adenoviral vector can comprise approximatelydifferent or equal amounts of the genome of each of the two or moredifferent adenovirus serotypes. For example, when the chimericadenoviral vector genome is comprised of the genomes of two differentadenovirus serotypes, the chimeric adenoviral vector genome preferablycomprises no more than about 99.9% (e.g., no more than about 99%, nomore than about 98%, no more than about 95%, no more than about 85%, nomore than about 80%, no more than about 75%, no more than about 60%, nomore than about 65%, or no more than about 50%) of the genome of one ofthe adenovirus serotypes, with the remainder of the chimeric adenovirusgenome being derived from the genome of the other adenovirus serotype.

The adenoviral vector can be replication-competent, conditionallyreplication-competent, or replication-deficient.

A replication-competent adenoviral vector can replicate in typical hostcells, i.e., cells typically capable of being infected by an adenovirus.A replication-competent adenoviral vector can have one or more mutationsas compared to a wild-type adenovirus (e.g., one or more deletions,insertions, and/or substitutions) in the adenoviral genome that do notinhibit viral replication in host cells. For example, the adenoviralvector can have a partial or entire deletion of the adenoviral earlyregion known as the E3 region, which is not essential for propagation ofthe adenoviral vector genome.

A conditionally-replicating adenoviral vector is an adenoviral vectorthat has been engineered to replicate under pre-determined conditions.For example, replication-essential gene functions, e.g., gene functionsencoded by the adenoviral early regions, can be operably linked to aninducible, repressible, or tissue-specific transcription controlsequence, e.g., a promoter. In such an embodiment, replication requiresthe presence or absence of specific factors that interact with thetranscription control sequence. Conditionally-replicating adenoviralvectors are further described in U.S. Pat. No. 5,998,205.

A replication-deficient adenoviral vector is an adenoviral vector thatrequires complementation of one or more gene functions or regions of theadenoviral genome that are required for replication, as a result of, forexample, a deficiency in one or more replication-essential gene functionor regions, such that the adenoviral vector does not replicate intypical host cells, especially those in a human to be infected by theadenoviral vector.

A deficiency in a gene function or genomic region, as used herein, isdefined as a disruption (e.g., deletion) of sufficient genetic materialof the adenoviral genome to obliterate or impair the function of thegene (e.g., such that the function of the gene product is reduced by atleast about 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, or 50-fold) whosenucleic acid sequence was disrupted (e.g., deleted) in whole or in part.Deletion of an entire gene region often is not required for disruptionof a replication-essential gene function. However, for the purpose ofproviding sufficient space in the adenoviral genome for one or moretransgenes, removal of a majority of one or more gene regions may bedesirable. While deletion of genetic material is preferred, mutation ofgenetic material by addition or substitution also is appropriate fordisrupting gene function. Replication-essential gene functions are thosegene functions that are required for adenovirus replication (e.g.,propagation) and are encoded by, for example, the adenoviral earlyregions (e.g., the E1, E2, and E4 regions), late regions (e.g., the L1,L2, L3, L4, and L5 regions), genes involved in viral packaging (e.g.,the IVa2 gene), and virus-associated RNAs (e.g., VA-RNA-1 and/orVA-RNA-2).

Whether the adenoviral vector is replication-competent orreplication-deficient, the adenoviral vector retains at least a portionof the adenoviral genome. The adenoviral vector can comprise any portionof the adenoviral genome, including protein coding and non-proteincoding regions. Desirably, the adenoviral vector comprises at least onenucleic acid sequence that encodes an adenovirus protein. The adenoviralvector can comprise a nucleic acid sequence that encodes any suitableadenovirus protein, such as, for example, a protein encoded by any oneof the early region genes (i.e., E1A, E1B, E2A, E2B, E3, and/or E4regions), or a protein encoded by any one of the late region genes,which encode the virus structural proteins (i.e., L1, L2, L3, L4, and L5regions).

Preferably, the adenoviral vector is replication-deficient, such thatthe replication-deficient adenoviral vector requires complementation ofat least one replication-essential gene function of one or more regionsof the adenoviral genome for propagation (e.g., to form adenoviralvector particles).

The replication-deficient adenoviral vector can be modified in anysuitable manner to cause the deficiencies in the one or morereplication-essential gene functions in one or more regions of theadenoviral genome for propagation. The complementation of thedeficiencies in the one or more replication-essential gene functions ofone or more regions of the adenoviral genome refers to the use ofexogenous means to provide the deficient replication-essential genefunctions. Such complementation can be effected in any suitable manner,for example, by using complementing cells and/or exogenous DNA (e.g.,helper adenovirus) encoding the disrupted replication-essential genefunctions.

The adenoviral vector can be deficient in one or morereplication-essential gene functions of only the early regions (i.e.,E1-E4 regions) of the adenoviral genome, only the late regions (i.e.,L1-L5 regions) of the adenoviral genome, both the early and late regionsof the adenoviral genome, or all adenoviral genes (i.e., a high capacityadenovector (HC-Ad)). See Morsy et al., Proc. Natl. Acad. Sci. USA, 95:965-976 (1998); Chen et al., Proc. Natl. Acad. Sci. USA, 94: 1645-1650(1997); and Kochanek et al., Hum. Gene Ther., 10: 2451-2459 (1999).Examples of replication-deficient adenoviral vectors are disclosed inU.S. Pat. Nos. 5,837,511; 5,851,806; 5,994,106; 6,127,175; 6,482,616;and 7,195,896, and International Patent Application Publications WO1994/028152, WO 1995/002697, WO 1995/016772, WO 1995/034671, WO1996/022378, WO 1997/012986, WO 1997/021826, and WO 2003/022311.

The early regions of the adenoviral genome include the E1, E2, E3, andE4 regions. The E1 region comprises the E1A and E1B subregions, and oneor more deficiencies in replication-essential gene functions in the E1region can include one or more deficiencies in replication-essentialgene functions in either or both of the E1A and E1B subregions, therebyrequiring complementation of the E1A subregion and/or the E1B subregionof the adenoviral genome for the adenoviral vector to propagate (e.g.,to form adenoviral vector particles). The E2 region comprises the E2Aand E2B subregions, and one or more deficiencies inreplication-essential gene functions in the E2 region can include one ormore deficiencies in replication-essential gene functions in either orboth of the E2A and E2B subregions, thereby requiring complementation ofthe E2A subregion and/or the E2B subregion of the adenoviral genome forthe adenoviral vector to propagate (e.g., to form adenoviral vectorparticles).

The E3 region does not include any replication-essential gene functions,such that a deletion of the E3 region in part or in whole does notrequire complementation of any gene functions in the E3 region for theadenoviral vector to propagate (e.g., to form adenoviral vectorparticles). In the context of the invention, the E3 region is defined asthe region that initiates with the open reading frame that encodes aprotein with high homology to the 12.5K protein from the E3 region ofhuman adenovirus 5 (NCBI reference sequence AP_000218) and ends with theopen reading frame that encodes a protein with high homology to the14.7K protein from the E3 region of human adenovirus 5 (NCBI referencesequence AP_000224.1). The E3 region may be deleted in whole or in part,or retained in whole or in part. The size of the deletion may betailored so as to retain an adenoviral vector whose genome closelymatches the optimum genome packaging size. A larger deletion willaccommodate the insertion of larger heterologous nucleic acid sequencesin the adenoviral genome. In one embodiment of the invention, the L4polyadenylation signal sequences, which reside in the E3 region, areretained.

The E4 region comprises multiple open reading frames (ORFs). Anadenoviral vector with a deletion of all of the open reading frames ofthe E4 region except ORF6, and in some cases ORF3, does not requirecomplementation of any gene functions in the E4 region for theadenoviral vector to propagate (e.g., to form adenoviral vectorparticles). Conversely, an adenoviral vector with a disruption ordeletion of ORF6, and in some cases ORF3, of the E4 region (e.g., with adeficiency in a replication-essential gene function based in ORF6 and/orORF3 of the E4 region), with or without a disruption or deletion of anyof the other open reading frames of the E4 region or the native E4promoter, polyadenylation sequence, and/or the right-side invertedterminal repeat (ITR), requires complementation of the E4 region(specifically, of ORF6 and/or ORF3 of the E4 region) for the adenoviralvector to propagate (e.g., to form adenoviral vector particles). Thelate regions of the adenoviral genome include the L1, L2, L3, L4, and L5regions. The adenoviral vector also can have a mutation in the majorlate promoter (MLP), as discussed in International Patent ApplicationPublication WO 2000/000628, which can render the adenoviral vectorreplication-deficient if desired.

The one or more regions of the adenoviral genome that contain one ormore deficiencies in replication-essential gene functions desirably areone or more early regions of the adenoviral genome, i.e., the E1, E2,and/or E4 regions, optionally with the deletion in part or in whole ofthe E3 region.

The replication-deficient adenoviral vector also can have one or moremutations as compared to a wild-type adenovirus (e.g., one or moredeletions, insertions, and/or substitutions) in the adenoviral genomethat do not inhibit viral replication in host cells. Thus, in additionto one or more deficiencies in replication-essential gene functions, theadenoviral vector can be deficient in other respects that are notreplication-essential. For example, the adenoviral vector can have apartial or entire deletion of the adenoviral early region known as theE3 region, which, as discussed herein, is not essential for propagationof the adenoviral genome.

In one embodiment, the adenoviral vector is replication-deficient andrequires, at most, complementation of the E1 region or the E4 region ofthe adenoviral genome, for propagation (e.g., to form adenoviral vectorparticles). Thus, the replication-deficient adenoviral vector requirescomplementation of at least one replication-essential gene function ofthe E1A subregion and/or the E1B region of the adenoviral genome(denoted an E1-deficient adenoviral vector) or the E4 region of theadenoviral genome (denoted an E4-deficient adenoviral vector) forpropagation (e.g., to form adenoviral vector particles). The adenoviralvector can be deficient in at least one replication-essential genefunction (desirably all replication-essential gene functions) of the E1region of the adenoviral genome and at least one gene function of thenonessential E3 region of the adenoviral genome (denoted anE1/E3-deficient adenoviral vector). The adenoviral vector can bedeficient in at least one replication-essential gene function (desirablyall replication-essential gene functions) of the E4 region of theadenoviral genome and at least one gene function of the nonessential E3region of the adenoviral genome (denoted an E3/E4-deficient adenoviralvector).

In one embodiment, the adenoviral vector is replication-deficient andrequires, at most, complementation of the E2 region, preferably the E2Asubregion, of the adenoviral genome, for propagation (e.g., to formadenoviral vector particles). Thus, the replication-deficient adenoviralvector requires complementation of at least one replication-essentialgene function of the E2A subregion of the adenoviral genome (denoted anE2A-deficient adenoviral vector) for propagation (e.g., to formadenoviral vector particles). The adenoviral vector can be deficient inat least one replication-essential gene function (desirably allreplication-essential gene functions) of the E2A region of theadenoviral genome and at least one gene function of the nonessential E3region of the adenoviral genome (denoted an E2A/E3-deficient adenoviralvector).

In one embodiment, the adenoviral vector is replication-deficient andrequires, at most, complementation of the E1 and E4 regions of theadenoviral genome for propagation (e.g., to form adenoviral vectorparticles). Thus, the replication-deficient adenoviral vector requirescomplementation of at least one replication-essential gene function ofboth the E1 and E4 regions of the adenoviral genome (denoted anE1/E4-deficient adenoviral vector) for propagation (e.g., to formadenoviral vector particles). The adenoviral vector can be deficient inat least one replication-essential gene function (desirably allreplication-essential gene functions) of the E1 region of the adenoviralgenome, at least one replication-essential gene function of the E4region of the adenoviral genome, and at least one gene function of thenonessential E3 region of the adenoviral genome (denoted anE1/E3/E4-deficient adenoviral vector). The adenoviral vector preferablyrequires, at most, complementation of the E1 region of the adenoviralgenome for propagation, and does not require complementation of anyother deficiency of the adenoviral genome for propagation. Morepreferably, the adenoviral vector requires, at most, complementation ofthe E1 and E4 regions of the adenoviral genome for propagation, and doesnot require complementation of any other deficiency of the adenoviralgenome for propagation.

The adenoviral vector, when deficient in multiple replication-essentialgene functions of the adenoviral genome (e.g., an E1/E4-deficientadenoviral vector), can include a spacer sequence to provide viralgrowth in a complementing cell line similar to that achieved byadenoviruses or adenoviral vectors deficient in a singlereplication-essential gene function (e.g., an E1-deficient adenoviralvector). The spacer sequence can contain any nucleotide sequence orsequences which are of a desired length, such as sequences at leastabout 15 base pairs (e.g., between about 15 nucleotides and about 12,000nucleotides), preferably about 100 nucleotides to about 10,000nucleotides, more preferably about 500 nucleotides to about 8,000nucleotides, even more preferably about 1,500 nucleotides to about 6,000nucleotides, and most preferably about 2,000 to about 3,000 nucleotidesin length, or a range defined by any two of the foregoing values. Thespacer sequence can be coding or non-coding and native or non-nativewith respect to the adenoviral genome, but does not restore thereplication-essential function to the deficient region. The spacer alsocan contain an expression cassette. More preferably, the spacercomprises a polyadenylation sequence and/or a gene that is non-nativewith respect to the adenoviral vector. The use of a spacer in anadenoviral vector is further described in, for example, U.S. Pat. No.5,851,806 and International Patent Application Publication WO1997/021826.

By removing all or part of the adenoviral genome, for example, the E1,E3, and E4 regions of the adenoviral genome, the resulting adenoviralvector is able to accept inserts of exogenous nucleic acid sequenceswhile retaining the ability to be packaged into adenoviral capsids. Anexogenous nucleic acid sequence can be inserted at any position in theadenoviral genome so long as insertion in the position allows for theformation of the adenoviral vector particle. The exogenous nucleic acidsequence (i.e., a nucleic acid sequence encoding a Plasmodium antigen)preferably is positioned in the E1 region, the E3 region, or the E4region of the adenoviral genome.

The replication-deficient adenoviral vector of the invention can beproduced in complementing cell lines that provide gene functions notpresent in the replication-deficient adenoviral vector, but required forviral propagation, at appropriate levels in order to generate hightiters of viral vector stock. Such complementing cell lines are knownand include, but are not limited to, 293 cells (described in, e.g.,Graham et al., J. Gen. Virol., 36: 59-72 (1977)), PER.C6 cells(described in, e.g., International Patent Application Publication WO1997/000326, and U.S. Pat. Nos. 5,994,128 and 6,033,908), and 293-ORF6cells (described in, e.g., International Patent Application PublicationWO 1995/034671 and Brough et al., J Virol., 71: 9206-9213 (1997)). Othersuitable complementing cell lines to produce the replication-deficientadenoviral vector of the invention include complementing cells that havebeen generated to propagate adenoviral vectors encoding transgenes whoseexpression inhibits viral growth in host cells (see, e.g., U.S. PatentApplication Publication 2008/0233650). Additional suitable complementingcells are described in, for example, U.S. Pat. Nos. 6,677,156 and6,682,929, and International Patent Application Publication WO2003/020879. In some instances, the cellular genome need not comprisenucleic acid sequences, the gene products of which complement for all ofthe deficiencies of a replication-deficient adenoviral vector. One ormore replication-essential gene functions lacking in areplication-deficient adenoviral vector can be supplied by a helpervirus, e.g., an adenoviral vector that supplies in trans one or moreessential gene functions required for replication of thereplication-deficient adenoviral vector. Alternatively, the adenoviralvector can comprise a non-native replication-essential gene thatcomplements for the one or more replication-essential gene functionslacking in the inventive replication-deficient adenoviral vector. Forexample, an E1/E4-deficient adenoviral vector can be engineered tocontain a nucleic acid sequence encoding E4 ORF6 that is obtained orderived from a different adenovirus (e.g., an adenovirus of a differentserotype than the inventive adenoviral vector, or an adenovirus of adifferent species than the inventive adenoviral vector).

In addition to the one or more nucleic acid sequences encodingPlasmodium antigens, the adenoviral vector preferably comprisesexpression control sequences, such as promoters, enhancers,polyadenylation signals, transcription terminators, internal ribosomeentry sites (IRES), and the like, that provide for the expression of thenucleic acid sequence in a host cell. Exemplary expression controlsequences are known in the art and are described in, for example,Goeddel, Gene Expression Technology: Methods in Enzymology, Vol. 185,Academic Press, San Diego, Calif. (1990). Ideally, the Plasmodiumantigen-encoding nucleic acid sequence is operably linked to a promoterand a polyadenylation sequence. The promoter desirably is a constitutiveor inducible promoter, preferably a constitutive promoter. A largenumber of promoters, including constitutive, inducible, and repressiblepromoters, from a variety of different sources are well known in theart. Representative sources of promoters include for example, virus,mammal, insect, plant, yeast, and bacteria, and suitable promoters fromthese sources are readily available, or can be made synthetically, basedon sequences publicly available, for example, from depositories such asthe ATCC as well as other commercial or individual sources. Promoterscan be unidirectional (i.e., initiate transcription in one direction) orbi-directional (i.e., initiate transcription in either a 3′ or 5′direction). Non-limiting examples of promoters include, for example, theT7 bacterial expression system, pBAD (araA) bacterial expression system,the cytomegalovirus (CMV) promoter (human or mouse), the β-actinpromoter (human or chicken), the EF1-α promoter, the ubiquitin promoter,the SV40 promoter, and the Rous Sarcoma Virus promoter. Induciblepromoters include, for example, the Tet system (U.S. Pat. Nos. 5,464,758and 5,814,618), the Ecdysone inducible system (No et al., Proc. Natl.Acad. Sci., 93: 3346-3351 (1996)), the T-REx™ system (Invitrogen,Carlsbad, Calif.), LACSWITCH™ system (Stratagene, San Diego, Calif.),and the Cre-ERT tamoxifen inducible recombinase system (Indra et al.,Nuc. Acid. Res., 27: 4324-4327 (1999); Nuc. Acid. Res., 28: e99 (2000);U.S. Pat. No. 7,112,715; and Kramer & Fussenegger, Methods Mol. Biol.,308: 123-144 (2005)).

A promoter can be selected by matching its particular pattern ofactivity with the desired pattern and level of expression of anantigen(s). For example, the adenoviral vector can comprise two or morenucleic acid sequences that encode the same or different antigens andare operably linked to different promoters displaying distinctexpression profiles. In this regard, a first promoter can be selected tomediate an initial peak of antigen production, thereby priming theimmune system against an encoded antigen. A second promoter can beselected to drive production of the same or different antigen such thatexpression peaks several days after that of the first promoter, thereby“boosting” the immune system against the antigen. Alternatively, ahybrid promoter can be constructed which combines the desirable aspectsof multiple promoters. For example, a CMV-Rous Sarcoma Virus hybridpromoter combining the CMV promoter's initial rush of activity with theRous Sarcoma Virus promoter's high maintenance level of activity can beemployed. In that antigens can be toxic to eukaryotic cells, it may beadvantageous to modify the promoter to decrease activity incomplementing cell lines used to propagate the adenoviral vector.

To optimize protein production, preferably the Plasmodiumantigen-encoding nucleic acid sequence further comprises apolyadenylation site following the coding sequence. Any suitablepolyadenylation sequence can be used, including a synthetic optimizedsequence, as well as the polyadenylation sequence of BGH (Bovine GrowthHormone), polyoma virus, TK (Thymidine Kinase), EBV (Epstein BarrVirus), mouse globin D protein, and the papillomaviruses, includinghuman papillomaviruses and BPV (Bovine Papilloma Virus). A preferredpolyadenylation sequence is the SV40 (Simian Virus-40) polyadenylationsequence. Also, preferably all the proper transcription signals (andtranslation signals, where appropriate) are correctly arranged such thatthe nucleic acid sequence is properly expressed in the cells into whichit is introduced. If desired, the nucleic acid sequence also canincorporate splice sites (i.e., splice acceptor and splice donor sites)to facilitate mRNA production.

If the nucleic acid sequence encodes a processed or secreted protein orpeptide, or a protein that acts intracellularly, preferably the nucleicacid sequence further comprises the appropriate sequences forprocessing, secretion, intracellular localization, and the like. Thenucleic acid sequence can be operably linked to a signal sequence, whichtargets a protein to cellular machinery for secretion. Appropriatesignal sequences include, but are not limited to, leader sequences forimmunoglobulin heavy chains and cytokines (see, for example, Ladunga etal., Current Opinions in Biotechnology, 11: 13-18 (2000)). Other proteinmodifications can be required to secrete a protein from a host cell,which can be determined using routine laboratory techniques. Preparingexpression constructs encoding antigens and signal sequences is furtherdescribed in, for example, U.S. Pat. No. 6,500,641. Methods of secretingnon-secretable proteins are further described in, for example, U.S. Pat.No. 6,472,176, and International Patent Application Publication WO2002/048377.

Plasmodium antigens encoded by the one or more nucleic acid sequencescan be modified to attach or incorporate the antigen on a host cellsurface. In this respect, the antigen can comprise a membrane anchor,such as a gpi-anchor, for conjugation onto a cell surface. Atransmembrane domain can be fused to the antigen to incorporate aterminus of the antigen protein into the cell membrane. Other strategiesfor displaying peptides on a cell surface are known in the art and areappropriate for use in the context of the invention.

The composition is a physiologically acceptable (e.g., pharmaceuticallyacceptable) composition, which comprises a carrier, preferably aphysiologically (e.g., pharmaceutically) acceptable carrier. Anysuitable carrier can be used within the context of the invention, andsuch carriers are well known in the art. The choice of carrier will bedetermined, in part, by the particular use of the composition (e.g.,administration to an animal) and the particular method used toadminister the composition. The composition optionally can be sterile.

Suitable compositions include aqueous and non-aqueous isotonic sterilesolutions, which can contain anti-oxidants, buffers, and bacteriostats,and aqueous and non-aqueous sterile suspensions that can includesuspending agents, solubilizers, thickening agents, stabilizers, andpreservatives. The composition can be presented in unit-dose ormulti-dose sealed containers, such as ampules and vials, and can bestored in a freeze-dried (lyophilized) condition requiring only theaddition of the sterile liquid carrier, for example, water, immediatelyprior to use. Extemporaneous solutions and suspensions can be preparedfrom sterile powders, granules, and tablets. Preferably, the carrier isa buffered saline solution. The composition can be generated inaccordance with conventional techniques described in, e.g., Remington:The Science and Practice of Pharmacy, 21^(st) Edition, LippincottWilliams & Wilkins, Philadelphia, Pa. (2001).

When the composition comprises an adenoviral vector, the compositionpreferably is free of replication-competent adenovirus. In addition, thecomposition preferably is formulated to protect the adenoviral vectorfrom damage prior to administration. For example, the composition can beformulated to reduce loss of the adenoviral vector on devices used toprepare, store, or administer the adenoviral vector, such as glassware,syringes, or needles. The composition can be formulated to decrease thelight sensitivity and/or temperature sensitivity of the adenoviralvector. To this end, the composition preferably comprises apharmaceutically acceptable liquid carrier, such as, for example, thosedescribed above, and a stabilizing agent selected from the groupconsisting of polysorbate 80, L-arginine, polyvinylpyrrolidone,trehalose, and combinations thereof. Use of such a composition willextend the shelf life of the adenoviral vector, and facilitate itsadministration. Formulations for adenoviral vector-containingcompositions are further described in, for example, U.S. Pat. No.6,225,289, U.S. Pat. No. 6,514,943, and International Patent ApplicationPublication WO 2000/034444.

The composition also can be formulated to enhance transductionefficiency of the composition. In addition, one of ordinary skill in theart will appreciate that the isolated Plasmodium polypeptides and/ornucleic acid sequences can be present in a composition with othertherapeutic or biologically-active agents. For example, factors thatcontrol inflammation, such as ibuprofen or steroids, can be part of thecomposition to reduce swelling and inflammation associated with in vivoadministration of the composition. Antibiotics, i.e., microbicides andfungicides, can be present to treat existing infection and/or reduce therisk of future infection, such as infection associated with genetransfer procedures.

The invention further provides a method of inducing a protective immuneresponse against a Plasmodium parasite in a mammal. The method comprisesadministering to the mammal the inventive composition comprisingisolated Plasmodium polypeptides or nucleic acid sequences, whereupon aprotective immune response against a Plasmodium parasite in the mammalis induced. When the composition comprises one or more Plasmodiumnucleic acid sequences, the one or more nucleic acid sequences encodingthe Plasmodium polypeptides are expressed in the mammal to produce thePlasmodium polypeptide. In accordance with the invention, thecomposition is administered to a mammal, most preferably a human. Thehuman preferably is in a population that has a high risk of acquiringPlasmodium parasites. Such high-risk populations include residents of,and travelers to, parts of Africa, Asia, the Middle East, Central andSouth America, Hispaniola, and Oceania, as well as military personneldeployed to these areas.

The immune response can be directed against any Plasmodium species thatinfects a particular mammal, such as those described herein. Preferably,the mammal is a human and the immune response is directed against ahuman-infecting Plasmodium species. Most preferably, the immune responseis directed against Plasmodium falciparum and/or Plasmodium vivax. Theimmune response can be a humoral immune response, a cell-mediated immuneresponse, or, desirably, a combination of humoral and cell-mediatedimmunity. Ideally, the immune response provides protection to theanimal, typically a mammal such as a human, upon subsequent challengewith Plasmodium. The inventive method also can be used for antibodyproduction and harvesting.

To enhance the immune response generated against a Plasmodium antigen,the composition also can comprise an immune stimulator, or a nucleicacid sequence that encodes an immune stimulator. Immune stimulators alsoare referred to in the art as “adjuvants,” and include, for example,cytokines, chemokines, or chaperones. Cytokines include, for example,Macrophage Colony Stimulating Factor (e.g., GM-CSF), Interferon Alpha(IFN-α), Interferon Beta (IFN-β), Interferon Gamma (IFN-γ), interleukins(IL-1, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-15, IL-16,and IL-18), the TNF family of proteins, Intercellular AdhesionMolecule-1 (ICAM-1), Lymphocyte Function-Associated antigen-3 (LFA-3),B7-1, B7-2, FMS-related tyrosine kinase 3 ligand, (Flt3L), vasoactiveintestinal peptide (VIP), and CD40 ligand. Chemokines include, forexample, B Cell-Attracting chemokine-1 (BCA-1), Fractalkine, MelanomaGrowth Stimulatory Activity (MGSA) protein, Hemofiltrate CC chemokine 1(HCC-1), Interleukin 8 (IL-8), Interferon-stimulated T-cell alphachemoattractant (I-TAC), Lymphotactin, Monocyte Chemotactic Protein 1(MCP-1), Monocyte Chemotactic Protein 3 (MCP-3), Monocyte ChemotacticProtein 4 (CP-4), Macrophage-Derived Chemokine (MDC), a macrophageinflammatory protein (MIP), Platelet Factor 4 (PF4), RANTES, BRAK,eotaxin, exodus 1-3, and the like. Chaperones include, for example, theheat shock proteins Hsp170, Hsc70, and Hsp40.

The composition ideally comprises a “therapeutically effective amount”of the isolated Plasmodium polypeptide or polypeptides. A“therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve a desiredtherapeutic result. The therapeutically effective amount may varyaccording to factors such as the disease state, age, sex, and weight ofthe individual, and the ability of the Plasmodium polypeptide to elicita desired response in the individual. For example, a therapeuticallyeffective amount of a Plasmodium polypeptide of the invention is anamount which ameliorates a malaria infection in a human.

Alternatively, the pharmacologic and/or physiologic effect may beprophylactic, i.e., the effect completely or partially prevents adisease or symptom thereof. In this respect, the inventive methodcomprises administering a “prophylactically effective amount” of thecomposition. A “prophylactically effective amount” refers to an amounteffective, at dosages and for periods of time necessary, to achieve adesired prophylactic result (e.g., prevention of Plasmodium infection oronset of malaria). For example, a prophylactically effective amount of aPlasmodium polypeptide of the invention is an amount which protects ahuman upon subsequent challenge with Plasmodium.

In embodiments where the composition comprises an adenoviral vectorcomprising one or more nucleic acid sequences that encode a Plasmodiumpolypeptide, the composition comprises a “therapeutically effectiveamount” of the adenoviral vector, i.e., a dose of adenoviral vectorwhich provokes a desired immune response in the mammal. Desirably, asingle dose of adenoviral vector comprises about 1×10⁵ or more particles(which also are referred to as particle units (pu)) of the adenoviralvector, e.g., about 1×10⁶ or more particles, about 1×10⁷ or moreparticles, about 1×10⁸ or more particles, about 1×10⁹ or more particles,or about 3×10⁹ or more particles of the adenoviral vector.Alternatively, or in addition, a single dose of adenoviral vectorcomprises about 3×10¹⁴ particles or less of the adenoviral vector, e.g.,about 1×10¹³ particles or less, about 1×10¹² particles or less, about3×10¹¹ particles or less, about 1×10¹¹ particles or less, about 1×10¹⁰particles or less, or about 1×10⁹ particles or less of the adenoviralvector. Thus, a single dose of adenoviral vector can comprise a quantityof particles of the adenoviral vector in a range defined by any two ofthe aforementioned values. For example, a single dose of adenoviralvector can comprise 1×10⁵-1×10¹⁴ particles, 1×10⁷-1×10¹² particles,1×10⁸-1×10¹¹ particles, 3×10⁸-3×10¹¹ particles, 1×10⁹-1×10¹² particles,1×10⁹-1×10¹¹ particles, 1×10⁹-1×10¹⁰ particles, or 1×10¹⁰-1×10¹²particles, of the adenoviral vector. In other words, a single dose ofadenoviral vector can comprise, for example, about 1×10⁶ pu, 2×10⁶ pu,4×10⁶ pu, 1×10⁷ pu, 2×10⁷ pu, 4×10⁷ pu, 1×10⁸ pu, 2×10⁸ pu, 3×10⁸ pu,4×10⁸ pu, 1×10⁹ pu, 2×10⁹ pu, 3×10⁹ pu, 4×10⁹ pu, 1×10¹⁰ pu, 2×10¹⁰ pu,3×10¹⁰ pu, 4×10¹⁰ pu, 1×10¹¹ pu, 2×10¹¹ pu, 3×10¹¹ pu, 4×10¹¹ pu, 1×10¹²pu, 2×10¹² pu, 3×10¹² pu, or 4×10¹² pu of the adenoviral vector.

Administration of the inventive composition can be one component of amultistep regimen for inducing a protective immune response againstPlasmodium parasites (e.g., Plasmodium falciparum and/or Plasmodiumvivax) in a mammal. In this respect, the method of inducing a protectiveimmune response can further comprise administering to the mammal aboosting composition after administering the inventive composition tothe mammal. In this embodiment, therefore, the immune response is“primed” upon administration of the inventive composition, and is“boosted” upon administration of the boosting composition.Alternatively, the inventive method further comprises administering tothe mammal a priming composition to the mammal prior to administeringthe inventive composition to the mammal. In this embodiment, therefore,the immune response is “primed” upon administration of the primingcomposition, and is “boosted” upon administration of the inventivecomposition.

In one embodiment, the Plasmodium polypeptide(s) present in, or encodedby a vector present in, the priming composition and the boostingcomposition are desirably the same. For example, if the primingcomposition comprises a hypothetical Plasmodium “protein A,” then theboosting composition also comprises “protein A,” or comprises a vectorwhich encodes “protein A.” At least one of the priming composition orthe boosting composition desirably comprises an adenoviral vector thatcomprises a nucleic acid sequence encoding a Plasmodium antigen, whilethe other of the priming composition and the boosting composition cancomprise the inventive composition or a different effective agent,though desirably a gene transfer vector that comprises a nucleic acidsequence encoding a Plasmodium antigen. Any gene transfer vector can beemployed, including viral and non-viral gene transfer vectors. Examplesof suitable viral gene transfer vectors include, but are not limited to,retroviral vectors, adeno-associated virus vectors, vaccinia virusvectors, herpesvirus vectors, parainfluenza-RSV chimeric vectors(PIV-RSV), and adenoviral vectors. Examples of suitable non-viralvectors include, but are not limited to, plasmids, liposomes, andmolecular conjugates (e.g., transferrin). Preferably, the primingcomposition or the boosting composition comprises a plasmid or anadenoviral vector. Alternatively, an immune response can be primed orboosted by administration of a Plasmodium protein itself (e.g., any ofthe Plasmodium proteins described herein) with or without a suitableadjuvant (e.g., alum, QS-21, insulin-derived adjuvant, etc.), alive-attenuated Plasmodium parasite, and the like. When the primingcomposition or the boosting composition comprises an adenoviral vector,the adenoviral vector can be, or can be derived from, any adenovirusthat infects a human or non-human animal, such as those describedherein. In this respect, the priming composition or the boostingcomposition can comprise a human adenoviral vector (e.g., serotype 5,28, or 35), a simian adenoviral vector (such as described in, e.g.,International Patent Application Publication WO 2011/057254), or agorilla adenoviral vector. For example, a priming composition containinga human serotype 5 adenoviral vector can be administered to a human,followed by administration of a boosting composition containing theinventive composition comprising one or more isolated Plasmodiumpolypeptides described herein (i.e., a “heterologous” prime-boostregimen). Alternatively, a priming composition containing the inventivecomposition comprising one or more isolated Plasmodium polypeptidesdescribed herein can be administered to a human, followed byadministration of a boosting composition containing a human serotype 5adenoviral vector. In another embodiment, the priming compositioncontains an adenoviral vector of a first serotype comprising a nucleicacid sequence encoding a Plasmodium antigen, and the boostingcomposition contains an adenoviral vector of a different serotypecomprising a nucleic acid sequence encoding the same Plasmodium antigenas the first adenoviral vector. Alternatively, the priming compositioncan contain a plasmid that comprises a nucleic acid sequence encoding aPlasmodium antigen, and the boosting composition can contain theinventive composition comprising an adenoviral vector. In yet anotherembodiment, the inventive composition comprising one or more isolatedPlasmodium polypeptides can be administered to a human, followed by asecond administration of the same composition (i.e., a “homologous”prime-boost regimen). One of ordinary skill in the art will appreciatethat any combination of vectors encoding one or more Plasmodium antigensand/or Plasmodium polypeptides themselves can be employed as the primingand/or boosting compositions in conjunction with the inventivecomposition described herein.

Administration of the priming composition and the boosting compositioncan be separated by any suitable timeframe (e.g., at least any of about1 week, 2 weeks, 4 weeks, 8 weeks, 12 weeks, 16 weeks, 24 weeks, 52weeks, 2 years, and 5 years, or any range defined by any two of theforegoing values). The boosting composition preferably is administeredto a mammal (e.g., a human) at least about 2 weeks (e.g., at least anyof about 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks,10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 20weeks, 24 weeks, 28 weeks, 35 weeks, 40 weeks, 50 weeks, 52 weeks, 2years, and 5 years, or any range defined by any two of the foregoingvalues) following administration of the priming composition. More thanone dose of priming composition and/or boosting composition can beprovided in any suitable timeframe. The dose of the priming compositionand boosting composition administered to the mammal depends on a numberof factors, including the extent of any side-effects, the particularroute of administration, and the like.

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

EXAMPLE 1

This example demonstrates a method for screening and isolatingPlasmodium antigen sequences that provide protection against malariachallenge.

Adenovirus-based microarray (“adeno-array”) technology (see, e.g., U.S.Patent Application Publication 2010/0222234) was used forhigh-throughput discovery of pre-erythrocytic Plasmodium falciparumantigens using orthologs identified in a P. yoelii (Py) mouse model.Specifically, bioinformatics data mining using publicly availablegenomic and proteomic databases was performed to identify highlyexpressed P. yoelii pre-erythrocytic antigens. Based on expressionabundance data from microarray analysis and protein mass spectrometryanalysis by several research groups, 300 sporozoite stage and liverstage candidate P. yoelii genes with identifiable P. falciparumorthologs were prioritized for generation of an adeno-array.

The candidate P. yoelii genes were cloned into a high-level adenovirusvector-based expression cassette using high-throughput methodologies(such as those described in, e.g., U.S. Patent Application Publication2010/0222234) to produce an adeno-array. Specifically, PCR-amplifiedcandidate antigens genes were first cloned into a pCR8/GW/Topo cloningvector (Life Technologies, Carlsbad, Calif.). The P. yoelii genes werethen individually subcloned into a CMV expression cassette which residesin a plasmid containing the human adenovirus 5 genome in which the E1and E3 regions were deleted. The CMV expression cassette resides in theE1 region of this plasmid. Cloning was carried out using theattR1-CmR-ccB-attR2 lambda recombination technology (Life Technologies,Carlsbad, Calif.). To construct the adenoviral vectors, the adenoviralgenomes were liberated from the plasmid backbone and used to transfect293-ORF6 complementing cells (Brough et al., J. Virol., 71: 9206-9213(1997)).

A20/2J antigen presenting cells (APCs) were infected with adenoviralvectors expressing Py antigens from the adeno-array and used as targetsin an IFNγ assay to recall T-cell responses from mice immunized withPlasmodium yoelii-irradiated sporozoites. The infected APCs were thenincubated with splenocytes from mice immunized with known protectiveregimens of Radiation Attenuated Sporozoites (RAS), and antigen-specificCD8³⁰T cell responses were measured by Intercellular Cytokine Staining(ICS). Adenoviral vectors encoding the Py circumsporozoite protein(PyCSP) and lacking a transgene (AdNull) were used as controls. Newantigens that recalled relatively frequent IFNγ responses inRAS-immunized, but not in naïve mice, were identified. The putative geneproduct encoded by each of the P. yoelii antigens and theircorresponding amino acid SEQ ID NOs, as well as the SEQ ID NOs for thecorresponding P. falciparum and P. vivax orthologs, are set forth inTable 1.

TABLE 1 P. yoelii P. falciparum P. vivax (17XNL) (3D7) Ortholog (SaI-1)Ortholog SEQ ID SEQ ID SEQ ID Putative Gene Product NO NO NO putativeWD-40 repeat 122 149 176 protein serine 123 150 177 hydroxymethyl-transferase, mitochondrial precursor 49 kDa zinc finger 124 151 178protein hypothetical protein 98 99 100 60S ribosomal protein 49 50 51L6, putative Arabidopsis thaliana 24 25 26 At1g20220/T20H2_3- relatedtranslation elongation 34 35 36 factor EF-1, subunit alpha adenosinedeaminase 14 15 16 translation initiation 125 152 179 factor eIF-5Astress-induced protein 126 153 180 sti1-like protein ethylene-inducible116 117 118 protein hever DNA replication 127 154 181 licensing factormis5 pyruvate dehydrogenase 128 155 182 E1 alpha subunit ribosomalprotein 129 156 183 var1, putative asparagine-rich 130 157 184 protein,putative succinyl-coa ligase beta- 131 158 185 chain, hydrogenosomalprecursor Drosophila melanogaster 132 159 186 RE21692p, putativecyclophilin 133 160 187 hypothetical protein 134 161 188 Protein ofunknown 135 162 189 function, putative Homo sapiens RIKEN 136 163 190cDNA 1600015H11 gene-related translation initiation 63 64 65 factor if-2Leucine Rich Repeat, 137 164 191 putative hypothetical protein 138 165192 DnaJ homolog, putative 139 166 193 ADP-ribosylation factor 140 167194 GTPase-activating protein ubiquitin carboxyl- 74 75 76 terminalhydrolase isozyme 15 elongation factor 3 141 168 195 related proteinPFEF3-rl eukaryotic translation 142 169 196 initiation factor 3 39 kDasubunit hypothetical protein 90 91 93 putative protein 84 85 86 Rab1protein 143 170 197 peptidyl-prolyl cis-trans 144 171 198 isomerase,cyclophilin- type Ribosomal protein S7e 145 172 199 similar to RIKENcDNA 146 173 200 2010107D16 gene neurofilament protein H 147 174 201form H2 60S acidic ribosomal 148 175 protein P2 putative protein 213 214215 phosphatase 2C putative small nucleolar 216 217 218ribonucleoprotein gar1 26s protease regulatory 219 220 221 subunit 6a(tat-binding protein homolog 1) (tbp-1) Drosophila melanogaster 222 223224 CG1349 gene product putative ribosomal 225 226 227 protein S19eglycyl-tRNA synthetase 228 229 230 transketolase 231 232 233asparaginyl-tRNA 234 235 236 synthetase putative acyl carrier 237 238239 protein putative calcyclin 240 241 242 binding protein putativeglycerol kinase 243 244 245 elongation factor 2 246 247 248 hexokinase249 250 251 pyruvate dehydrogenase 252 253 254 E1 beta subunit U43539hepatocyte 255 256 257 erythrocyte protein 17 kDa protein kinase domain258 259 260 merozoite surface protein 261 262 263 1 precursor merozoitesurface protein 264 265, 266, 267 268 7 precursor glutathiones-transferase 269 N/A 270 S-adenosylmethionine 271 272 273 synthetase

Antigens that recalled the most robust T cell responses were selectedand their capacity to protect mice from a P. yoelii sporozoite challengewas tested. In this regard, 100 μg of plasmid vector expressing each ofthe above Py antigens were injected into mice at the start (Day 0) ofthe experiment, and 6 weeks later, animals were boosted by intramuscular(IM) injection of adenoviral vectors expressing the same antigens at1×10¹⁰ particles per mouse. Immunized mice were challenged with 300 or600 live sporozoites two weeks after boost. Tail vein bleeds wereperformed from 6 to 14 days after live sporozoite challenge and bloodfilms were prepared for analysis of blood-stage parasitemia. Outbred CD1mice were immunized with a DNA prime-adenovirus boost regimen andsterile protection was measured following sporozoite challenge as setforth in Table 2.

TABLE 2 Sterile protection Antigen SEQ ID NO # Protected/# challenged %protected PyCSP 53/112 47% GV0032 24 16/41  39% GV0041 14 10/42  24%GV0033 34 6/28 21% GV0043 125 1/14  7% GV0074 213 4/14 29% GV0176 747/28 25% GV0196 90 7/28 25% GV0004 123 3/14 21% GV0013 124 3/14 21%GV0199 84 6/28 21% GV0014 98 8/42 19% GV0139 216 3/14 21% GV0104 2711/14  7%

 control 11/112 10% AdNull  8/112  7%

The results of this example demonstrate the identification of newPlasmodium antigens that can induce a protective immune response againstmalaria in a mammal.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and “at least one” andsimilar referents in the context of describing the invention (especiallyin the context of the following claims) are to be construed to coverboth the singular and the plural, unless otherwise indicated herein orclearly contradicted by context. The use of the term “at least one”followed by a list of one or more items (for example, “at least one of Aand B”) is to be construed to mean one item selected from the listeditems (A or B) or any combination of two or more of the listed items (Aand B), unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

The invention claimed is:
 1. A method of inducing a protective immuneresponse against a Plasmodium parasite in a mammal, wherein the methodcomprises administering to the mammal a composition comprising apharmaceutically acceptable carrier and one or more isolatedpolypeptides, wherein each of the one or more isolated polypeptidescomprises: (a) an amino acid sequence comprising at least 20 contiguousamino acid residues of the sequence MKKDREPIDEDEMRITSTGRMTNYVNYGAKILG(SEQ ID NO: 20), (b) an amino acid sequence comprising at least 20contiguous amino acid residues of the sequenceKIKATGNAIGKAVTLAEIIKRRFKGLHQIT (SEQ ID NO: 21), or (c) an amino acidsequence comprising SEQ ID NO: 22, and wherein each of the one or moreisolated polypeptides induces a protective immune response againstPlasmodium falciparum and/or Plasmodium vivax in a mammal.
 2. The methodof claim 1, wherein the composition comprises an isolated polypeptidecomprising at least 20 contiguous amino acid residues of the sequenceMKKDREPIDEDEMRITSTGRMTNYVNYGAKILG (SEQ ID NO: 20).
 3. The method ofclaim 2, wherein the composition comprises an isolated polypeptidecomprising the amino acid sequence of SEQ ID NO:
 20. 4. The method ofclaim 1, wherein the composition comprises an isolated polypeptidecomprising at least 20 contiguous amino acid residues of the sequenceKIKATGNAIGKAVTLAEIIKRRFKGLHQIT (SEQ ID NO: 21).
 5. The method of claim4, wherein the composition comprises an isolated polypeptide comprisingthe amino acid sequence of SEQ ID NO:
 21. 6. The method of claim 1,wherein the composition comprises an isolated polypeptide comprising theamino acid sequence of SEQ ID NO:
 22. 7. The method of claim 6, whereinthe isolated polypeptide comprises the amino acid sequence ofKDAGYQPPLDEKYVKEMXPEEIVN (SEQ ID NO: 23), wherein X is a serine (S)residue or a threonine (T) residue.
 8. The method of claim 1, whereinthe composition comprises a polypeptide comprising the amino acidsequence of SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO:
 26. 9. Themethod of claim 1, wherein the mammal is a human.
 10. A method ofinducing a protective immune response against a Plasmodium parasite in amammal, wherein the method comprises administering to the mammal acomposition comprising a pharmaceutically acceptable carrier and one ormore isolated polypeptides, wherein each of the one or more isolatedpolypeptides comprises: (a) an amino acid sequence comprising at least20 contiguous amino acid residues of the sequenceMKKDREPIDEDEMRITSTGRMTNYVNYGAKILG (SEQ ID NO: 20), (b) an amino acidsequence comprising at least 20 contiguous amino acid residues of thesequence KIKATGNAIGKAVTLAEIIKRRFKGLHQIT (SEQ ID NO: 21), and (c) anamino acid sequence comprising SEQ ID NO: 22, and wherein each of theone or more isolated polypeptides induces a protective immune responseagainst Plasmodium falciparum and/or Plasmodium vivax in a mammal. 11.The method of claim 10, wherein the isolated polypeptide comprises theamino acid sequence of KDAGYQPPLDEKYVKEMXPEEIVN (SEQ ID NO: 23), whereinX is a serine (S) residue or a threonine (T) residue.
 12. The method ofclaim 10, wherein one or more isolated polypeptides comprises the aminoacid sequences of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO:
 22. 13.The method of claim 12, wherein the isolated polypeptide comprises theamino acid sequence of KDAGYQPPLDEKYVKEMXPEEIVN (SEQ ID NO: 23), whereinX is a serine (S) residue or a threonine (T) residue.
 14. The method ofclaim 13, wherein the mammal is a human.
 15. The method of claim 10,wherein the mammal is a human.
 16. The method of claim 11, wherein themammal is a human.
 17. The method of claim 12, wherein the mammal is ahuman.
 18. A method of inducing a protective immune response against aPlasmodium parasite in a mammal, wherein the method comprisesadministering to the mammal a composition comprising a pharmaceuticallyacceptable carrier and one or more polypeptides comprising the aminoacid sequence of SEQ ID NO: 24, wherein the Plasmodium parasite isPlasmodium falciparum or Plasmodium vivax.
 19. The method of claim 18,wherein the mammal is a human.
 20. A method of inducing a protectiveimmune response against a Plasmodium parasite in a mammal, wherein themethod comprises administering to the mammal a composition comprising apharmaceutically acceptable carrier and one or more polypeptidescomprising the amino acid sequence of SEQ ID NO: 25, wherein thePlasmodium parasite is Plasmodium falciparum or Plasmodium vivax. 21.The method of claim 20, wherein the mammal is a human.
 22. A method ofinducing a protective immune response against a Plasmodium parasite in amammal, wherein the method comprises administering to the mammal acomposition comprising a pharmaceutically acceptable carrier and one ormore polypeptides comprising the amino acid sequence of SEQ ID NO: 26,wherein the Plasmodium parasite is Plasmodium falciparum or Plasmodiumvivax.
 23. The method of claim 22, wherein the mammal is a human.